Growth differentiation factor 15 (GDF-15) constructs

ABSTRACT

Constructs comprising GDF15, and mutants thereof are provided. In various embodiments the constructs comprising GDF15, and mutants thereof, can be of use in the treatment or ameliorating a metabolic disorder. In various embodiments the metabolic disease or disorder is type 2 diabetes, obesity, dyslipidemia, elevated glucose levels, elevated insulin levels and diabetic nephropathy.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jul. 29, 2014, isnamed A-1850-WO-PCT_SL.txt and is 630,200 bytes in size.

FIELD OF THE INVENTION

The instant disclosure relates to monomers and multimers comprising apolypeptide comprising a GDF15 region.

BACKGROUND OF THE INVENTION

Growth differentiation factor 15 (GDF15) is a divergent member of theTGFβ superfamily. It is also called macrophage inhibitory cytokine 1(MIC1) (Bootcov M R, 1997, Proc Natl Acad Sci 94:11514-9), placentalbone morphogenetic factor (PLAB) (Hromas R 1997, Biochim Biophys Acta.1354:40-4), placental transforming growth factor beta (PTGFB) (Lawton LN 1997, Gene. 203:17-26), prostate derived factor (PDF) (Paralkar V M1998, J Biol Chem. 273:13760-7), and nonsteroidal anti-inflammatorydrug-activated gene (NAG-1) (Baek S J 2001, J Biol Chem. 276: 33384-92).

Human GDF15 gene is located on chromosome 19p13.2-13.1; rat GDF15 geneis located on chromosome 16; and mouse GDF15 gene is located onchromosome 8. The GDF15 open reading frames span two exons (Bottner M1999, Gene. 237:105-11 and NCBI). The mature GDF15 peptide shares lowhomology with other family members (Katoh M 2006, Int J Mol Med.17:951-5.).

GDF15 is synthesized as a large precursor protein that is cleaved at thedibasic cleavage site to release the carboxyterminal mature peptide. Themouse and rat GDF15 prepro-peptides both contain 303 amino acids. Humanfull-length precursor contains 308 amino acids. The rodent maturepeptides contain 115 amino acids after processing at the RGRR (SEQ IDNO:1) cleavage site. The human mature peptide contains 112 amino acidsafter processing at the RGRRRAR (SEQ ID NO:2) cleavage site. Humanmature GDF15 peptide shares 66.1% and 68.1% sequence similarity with ratand mouse mature GDF15 peptides (Bottner M 1999, Gene. 237:105-11;Bauskin A R 2000, EMBO J. 19:2212-20; NCBI). There is no glycosylationsite in the mature GDF15 peptide.

The mature GDF15 peptide contains the seven conserved cysteine residuesrequired for the formation of the cysteine knot motif (having threeintrachain disulfide bonds) and the single interchain disulfide bondthat are typical for TGFβ superfamily members. The mature GDF15 peptidefurther contains two additional cysteine residues that form a fourthintrachain disulfide bond. Biologically active GDF15 is a 25 KDhomodimer of the mature peptide covalently linked by one interchaindisulfide bond.

GDF15 circulating levels have been reported to be elevated in multiplepathological and physiological conditions, most notably pregnancy (MooreA G 2000. J Clin Endocrinol Metab 85: 4781-4788), β-thalassemia (Tanno T2007, Nat Med 13:1096-101; Zimmermann M B, 2008 Am J Clin Nutr88:1026-31), and congenital dyserythropoietic anemia (Tamary H 2008,Blood. 112:5241-4). GDF15 has also been linked to multiple biologicalactivities in literature reports. Studies of GDF15 knockout andtransgenic mice suggested that GDF15 may be protective againstischemic/reperfusion- or overload-induced heart injury (Kempf T, 2006,Circ Res. 98:351-60; Xu J, 2006, Circ Res. 98:342-50), protectiveagainst aging-associated motor neuron and sensory neuron loss (StrelauJ, 2009, J Neurosci. 29:13640-8), mildly protective against metabolicacidosis in kidney, and may cause cachexia in cancer patients (Johnen H2007 Nat Med. 11:1333-40). Many groups also have studied the role ofGDF15 in cell apoptosis and proliferation and reported controversialresults using different cell culture and xenograft models. Studies ontransgenic mice showed that GDF15 is protective against carcinogen orApc mutation induced neoplasia in intestine and lung (Baek S J 2006,Gastroenterology. 131:1553-60; Cekanova M 2009, Cancer Prev Res2:450-8).

SUMMARY OF THE INVENTION

Provided herein are fusion proteins comprising a GDF15 polypeptide or aGDF15 mutant polypeptide and an Fc domain.

In a one embodiment, the Fc domain comprises a sequence selected fromthe group consisting of SEQ ID NOs:16, 22, 28, 29, 33, 35, 38, 48, 85,91, 106, 132, 141, 148, 155, 162, 169, 176, 183, 192, 199, 206, 213,220, 227, 233, 236, 268, 275, 281, 282, 283, 284, 285, 286, 287, 288,289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301 and 302.In another embodiment, the GDF15 polypeptide or a GDF15 mutantpolypeptide comprises a sequence selected from the group consisting ofSEQ ID NOs:4, 8, 12, 25, 52 and 55. In another embodiment, the fusionprotein further comprises a polypeptide linker. In one embodiment, thepolypeptide linker has a sequence selected from the group consisting ofSEQ ID NOs:18, 30, 34, 40, 58, 61, 64, 69, 72, 75, 78, 113, 116, 119,122, 125, 128. In one embodiment, the fusion protein comprises two ormore Fc domains. In one embodiment, the fusion protein comprises two ormore polypeptide linkers. In one embodiment, the fusion proteincomprises a sequence selected from the group consisting of SEQ ID NOs:46, 24, 27, 32, 37, 20, 42, 50, 54, 57, 60, 63, 66, 68, 71, 74, 77, 82,84, 88, 93, 96, 98, 100, 102, 104, 108, 134, 137, 139, 143, 146, 150,153, 269, 272, 276, 279, 157, 160, 164, 167, 171, 174, 178, 181, 185,188, 194, 197, 201, 204, 208, 211, 215, 218, 222, 225, 229, 232, 233,238 and 240.

Also provided herein are dimers comprising (i) a first polypeptide chaincomprising one of the foregoing fusion proteins, and (ii) a secondpolypeptide chain comprising an Fc domain. In yet a further embodiment,the construct further comprises a sequence selected from the groupconsisting of SEQ ID NOs: 16, 22, 28, 29, 33, 35, 38, 48, 85, 91, 106,132, 141, 148, 155, 162, 169, 176, 183, 192, 199, 206, 213, 220, 227,233, 236, 268, 275, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290,291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301 and 302.

In one embodiment, the first and second polypeptide chains arenon-covalently associated. In another embodiment, the first and secondpolypeptide chains are covalently associated. In one embodiment, thefirst and second polypeptide chains are covalently associated viadisulfide bonds between their respective Fc domains. In anotherembodiment, the first and second polypeptide chains are associated byboth covalent and non-covalent interactions.

In a particular embodiment, a dimer is provided comprising: (a) twofusion proteins comprising the sequence of SEQ ID NO:46; (b) two fusionproteins comprising the sequence of SEQ ID NO:24; or (c) two fusionproteins comprising the sequence of SEQ ID NO:27

In a particular embodiment, a dimer is provided comprising (a) twofusion proteins comprising the sequence of SEQ ID NO:32; or (b) twofusion proteins comprising the sequence of SEQ ID NO:37;

In a particular embodiment, a dimer is provided comprising a firstpolypeptide chain comprising the sequence of SEQ ID NO:20 and a secondpolypeptide chain comprising the sequence of SEQ ID NO:17.

In a particular embodiment, a dimer is provided comprising (a) a firstpolypeptide chain comprising the sequence of SEQ ID NO:42 and a secondpolypeptide chain comprising the sequence of SEQ ID NO:39; (b) a firstpolypeptide chain comprising the sequence of SEQ ID NO:50 and a secondpolypeptide chain comprising the sequence of SEQ ID NO:47; (c) a firstpolypeptide chain comprising the sequence of SEQ ID NO:54 and a secondpolypeptide chain comprising the sequence of SEQ ID NO:47; (d) a firstpolypeptide chain comprising the sequence of SEQ ID NO:57 and a secondpolypeptide chain comprising the sequence of SEQ ID NO:47; (e) a firstpolypeptide chain comprising the sequence of SEQ ID NO:60 and a secondpolypeptide chain comprising the sequence of SEQ ID NO:47; (f) a firstpolypeptide chain comprising the sequence of SEQ ID NO:63 and a secondpolypeptide chain comprising the sequence of SEQ ID NO:47; (g) a firstpolypeptide chain comprising the sequence of SEQ ID NO:66 and a secondpolypeptide chain comprising the sequence of SEQ ID NO:47; (h) a firstpolypeptide chain comprising the sequence of SEQ ID NO:68 and a secondpolypeptide chain comprising the sequence of SEQ ID NO:47; (i) a firstpolypeptide chain comprising the sequence of SEQ ID NO:71 and a secondpolypeptide chain comprising the sequence of SEQ ID NO:47; (j) a firstpolypeptide chain comprising the sequence of SEQ ID NO:74 and a secondpolypeptide chain comprising the sequence of SEQ ID NO:47; (k) a firstpolypeptide chain comprising the sequence of SEQ ID NO:77 and a secondpolypeptide chain comprising the sequence of SEQ ID NO:47; (l) a firstpolypeptide chain comprising the sequence of SEQ ID NO:80 and a secondpolypeptide chain comprising the sequence of SEQ ID NO:47; (m) a firstpolypeptide chain comprising the sequence of SEQ ID NO:82 and a secondpolypeptide chain comprising the sequence of SEQ ID NO:47; or (n) afirst polypeptide chain comprising the sequence of SEQ ID NO:84 and asecond polypeptide chain comprising the sequence of SEQ ID NO:47.

In a particular embodiment, a dimer is provided comprising (a) a firstpolypeptide chain comprising the sequence of SEQ ID NO:88 and a secondpolypeptide chain comprising the sequence of SEQ ID NO:86; (b) a firstpolypeptide chain comprising the sequence of SEQ ID NO:93 and a secondpolypeptide chain comprising the sequence of SEQ ID NO:90; (c) a firstpolypeptide chain comprising the sequence of SEQ ID NO:96 and a secondpolypeptide chain comprising the sequence of SEQ ID NO:90; (d) a firstpolypeptide chain comprising the sequence of SEQ ID NO:98 and a secondpolypeptide chain comprising the sequence of SEQ ID NO:90; (e) a firstpolypeptide chain comprising the sequence of SEQ ID NO:100 and a secondpolypeptide chain comprising the sequence of SEQ ID NO:90; (f) a firstpolypeptide chain comprising the sequence of SEQ ID NO:102 and a secondpolypeptide chain comprising the sequence of SEQ ID NO:90; (g) a firstpolypeptide chain comprising the sequence of SEQ ID NO:104 and a secondpolypeptide chain comprising the sequence of SEQ ID NO:90; or (h) afirst polypeptide chain comprising the sequence of SEQ ID NO:108 and asecond polypeptide chain comprising the sequence of SEQ ID NO:105.

In a particular embodiment, a dimer is provided comprising (a) a firstpolypeptide chain comprising the sequence of SEQ ID NO:112 and a secondpolypeptide chain comprising the sequence of SEQ ID NO:12; (b) twopolypeptide chains, each comprising the sequence of SEQ ID NO:112; (c)two polypeptide chains, each comprising the sequence of SEQ ID NO:115;(d) two polypeptide chains, each comprising the sequence of SEQ IDNO:118; (e) two polypeptide chains, each comprising the sequence of SEQID NO:121; (f) two polypeptide chains, each comprising the sequence ofSEQ ID NO:124; (g) two polypeptide chains, each comprising the sequenceof SEQ ID NO:127; (h) two polypeptide chains, each comprising thesequence of SEQ ID NO:130; or (i) two polypeptide chains, eachcomprising the sequence of SEQ ID NO:242.

In a particular embodiment, a dimer is provided comprising: (a) a firstpolypeptide chain comprising the sequence of SEQ ID NO:134 and a secondpolypeptide chain comprising the sequence of SEQ ID NO:131; (b) a firstpolypeptide chain comprising the sequence of SEQ ID NO:137 and a secondpolypeptide chain comprising the sequence of SEQ ID NO:131; (c) a firstpolypeptide chain comprising the sequence of SEQ ID NO:139 and a secondpolypeptide chain comprising the sequence of SEQ ID NO:131; (d) a firstpolypeptide chain comprising the sequence of SEQ ID NO:143 and a secondpolypeptide chain comprising the sequence of SEQ ID NO:140; (e) a firstpolypeptide chain comprising the sequence of SEQ ID NO:146 and a secondpolypeptide chain comprising the sequence of SEQ ID NO:140; (f) a firstpolypeptide chain comprising the sequence of SEQ ID NO:150 and a secondpolypeptide chain comprising the sequence of SEQ ID NO:147; (g) a firstpolypeptide chain comprising the sequence of SEQ ID NO:153 and a secondpolypeptide chain comprising the sequence of SEQ ID NO:147; (h) a firstpolypeptide chain comprising the sequence of SEQ ID NO:269 and a secondpolypeptide chain comprising the sequence of SEQ ID NO:267; (i) a firstpolypeptide chain comprising the sequence of SEQ ID NO:272 and a secondpolypeptide chain comprising the sequence of SEQ ID NO:267; (j) a firstpolypeptide chain comprising the sequence of SEQ ID NO:276 and a secondpolypeptide chain comprising the sequence of SEQ ID NO:274; or (k) afirst polypeptide chain comprising the sequence of SEQ ID NO:279 and asecond polypeptide chain comprising the sequence of SEQ ID NO:274.

In a particular embodiment, a dimer is provided comprising: (a) a firstpolypeptide chain comprising the sequence of SEQ ID NO:157 and a secondpolypeptide chain comprising the sequence of SEQ ID NO:154; (b) a firstpolypeptide chain comprising the sequence of SEQ ID NO:160 and a secondpolypeptide chain comprising the sequence of SEQ ID NO:154; (c) a firstpolypeptide chain comprising the sequence of SEQ ID NO:164 and a secondpolypeptide chain comprising the sequence of SEQ ID NO:161; (d) a firstpolypeptide chain comprising the sequence of SEQ ID NO:167 and a secondpolypeptide chain comprising the sequence of SEQ ID NO:161.

In a particular embodiment, a dimer is provided comprising: (a) a firstpolypeptide chain comprising the sequence of SEQ ID NO:171 and a secondpolypeptide chain comprising the sequence of SEQ ID NO:168; or (b) afirst polypeptide chain comprising the sequence of SEQ ID NO:174 and asecond polypeptide chain comprising the sequence of SEQ ID NO:168

In a particular embodiment, a dimer is provided comprising: (a) a firstpolypeptide chain comprising the sequence of SEQ ID NO:178 and a secondpolypeptide chain comprising the sequence of SEQ ID NO:175; (b) a firstpolypeptide chain comprising the sequence of SEQ ID NO:181 and a secondpolypeptide chain comprising the sequence of SEQ ID NO:175; (c) a firstpolypeptide chain comprising the sequence of SEQ ID NO:185 and a secondpolypeptide chain comprising the sequence of SEQ ID NO:182; (d) a firstpolypeptide chain comprising the sequence of SEQ ID NO:188 and a secondpolypeptide chain comprising the sequence of SEQ ID NO:182; (e) a firstpolypeptide chain comprising the sequence of SEQ ID NO:194 and a secondpolypeptide chain comprising the sequence of SEQ ID NO:191; (f) a firstpolypeptide chain comprising the sequence of SEQ ID NO:197 and a secondpolypeptide chain comprising the sequence of SEQ ID NO:191; (g) a firstpolypeptide chain comprising the sequence of SEQ ID NO:201 and a secondpolypeptide chain comprising the sequence of SEQ ID NO:198; or (h) afirst polypeptide chain comprising the sequence of SEQ ID NO:204 and asecond polypeptide chain comprising the sequence of SEQ ID NO:198.

In a particular embodiment, a dimer is provided comprising: (a) a firstpolypeptide chain comprising the sequence of SEQ ID NO:208 and a secondpolypeptide chain comprising the sequence of SEQ ID NO:205; (b) a firstpolypeptide chain comprising the sequence of SEQ ID NO:211 and a secondpolypeptide chain comprising the sequence of SEQ ID NO:205; (c) a firstpolypeptide chain comprising the sequence of SEQ ID NO:215 and a secondpolypeptide chain comprising the sequence of SEQ ID NO:212; or (d) afirst polypeptide chain comprising the sequence of SEQ ID NO:218 and asecond polypeptide chain comprising the sequence of SEQ ID NO:212.

In a particular embodiment, a dimer is provided comprising: (a) a firstpolypeptide chain comprising the sequence of SEQ ID NO:222 and a secondpolypeptide chain comprising the sequence of SEQ ID NO:219; (b) a firstpolypeptide chain comprising the sequence of SEQ ID NO:225 and a secondpolypeptide chain comprising the sequence of SEQ ID NO:219; (c) a firstpolypeptide chain comprising the sequence of SEQ ID NO:229 and a secondpolypeptide chain comprising the sequence of SEQ ID NO:226; or (d) afirst polypeptide chain comprising the sequence of SEQ ID NO:232 and asecond polypeptide chain comprising the sequence of SEQ ID NO:226.

In a particular embodiment, a dimer is provided comprising: (a) twopolypeptide chains, each comprising the sequence of SEQ ID NO:235; (b)two polypeptide chains, each comprising the sequence of SEQ ID NO:238;or (c) two polypeptide chains, each comprising the sequence of SEQ IDNO:240.

In certain embodiments, a tetramer is provided, comprising (i) a firstdimer comprising one of the foregoing dimers and (ii) a second dimercomprising one of the foregoing dimers. In certain embodiments, thefirst polypeptide chain of the first dimer is linked to the firstpolypeptide chain of the second dimer via an interchain disulfide bondbetween their respective GDF15 regions.

In some embodiments, the dimer is not selected from the group comprisingDhCpmFc(−)-(G₄S)₄-GDF15:DhCpmFc(+), DhCpmFc(+)-(G₄S)₄-GDF15:DhCpmFc(−),DhCpmFc(−)-(G₄S)₄-GDF15(H6D):DhCpmFc(+),DhCpmFc(+)-(G₄S)₄-GDF15(H6D):DhCpmFc(−),DhCpmFc(+)-(G₄S)₄-GDF15(N3Q):DhCpmFc(−), DhCpmFc(+)-GDF15:DhCpmFc(−),DhCpmFc(+)-G₄-GDF15:DhCpmFc(−), DhCpmFc(+)-(G₄S)₂-GDF15:DhCpmFc(−),DhCpmFc(+)-(G₄Q)₄-GDF15:DhCpmFc(−),DhCpmFc(+)(L351C)-G₄-GDF15:DhCpmFc(−)(L351C),DhCpmFc(+)(S354C)-G₄-GDF15:DhCpmFc(−)(Y349C),CpmFc(−)-(G₄S)₄-GDF15:CpmFc(+), Fc-(G₄S)₈-Fc-GS(G₄S)₄-GDF15,Fc-(G₄S)₃-Fc-GS(G₄S)₄-GDF15 and Fc-(G₄S)₅-Fc-GS(G₄S)₄-GDF15.

In some embodiments, the dimer is not selected from the group consistingof DhCpmFc(−)-(G₄S)₄-GDF15:DhCpmFc(+),DhCpmFc(+)-(G₄S)₄-GDF15:DhCpmFc(−),DhCpmFc(−)-(G₄S)₄-GDF15(H6D):DhCpmFc(+),DhCpmFc(+)-(G₄S)₄-GDF15(H6D):DhCpmFc(−),DhCpmFc(+)-(G₄S)₄-GDF15(N3Q):DhCpmFc(−), DhCpmFc(+)-GDF15:DhCpmFc(−),DhCpmFc(+)-G₄-GDF15:DhCpmFc(−), DhCpmFc(+)-(G₄S)₂-GDF15:DhCpmFc(−),DhCpmFc(+)-(G₄Q)₄-GDF15:DhCpmFc(−),DhCpmFc(+)(L351C)-G₄-GDF15:DhCpmFc(−)(L351C) andDhCpmFc(+)(S354C)-G₄-GDF15:DhCpmFc(−)(Y349C).

In some embodiments, the fusion protein is not a selected from the groupconsisting of Fc-(G₄S)₈-Fc-GS(G₄S)₄-GDF15, Fc-(G₄S)₃-Fc-GS(G₄S)₄-GDF15and Fc-(G₄S)₅-Fc-GS(G₄S)₄-GDF15.

In some embodiments, the dimer is not selected from the group consistingof DhCpmFc(+)(L351C)-G₄-GDF15:DhCpmFc(−)(L351C) andDhCpmFc(+)(S354C)-G₄-GDF15:DhCpmFc(−)(Y349C).

In some embodiments, the dimer is notDhCpmFc(−)-(G₄S)₄-GDF15:DhCpmFc(+), DhCpmFc(+)-(G₄S)₄-GDF15:DhCpmFc(−),DhCpmFc(−)-(G₄S)₄-GDF15(H6D):DhCpmFc(+),DhCpmFc(+)-(G₄S)₄-GDF15(H6D):DhCpmFc(−),DhCpmFc(+)-(G₄S)₄-GDF15(N3Q):DhCpmFc(−), DhCpmFc(+)-GDF15:DhCpmFc(−),DhCpmFc(+)-G₄-GDF15:DhCpmFc(−), DhCpmFc(+)-(G₄S)₂-GDF15:DhCpmFc(−),DhCpmFc(+)-(G₄Q)₄-GDF15:DhCpmFc(−),DhCpmFc(+)(L351C)-G₄-GDF15:DhCpmFc(−)(L351C),DhCpmFc(+)(S354C)-G₄-GDF15:DhCpmFc(−)(Y349C),CpmFc(−)-(G₄S)₄-GDF15:CpmFc(+), Fc-(G₄S)₈-Fc-GS(G₄S)₄-GDF15,Fc-(G₄S)₃-Fc-GS(G₄S)₄-GDF15 or Fc-(G₄S)₅-Fc-GS(G₄S)₄-GDF15.

In some embodiments, the dimer is notDhCpmFc(−)-(G₄S)₄-GDF15:DhCpmFc(+), DhCpmFc(+)-(G₄S)₄-GDF15:DhCpmFc(−),DhCpmFc(−)-(G₄S)₄-GDF15(H6D):DhCpmFc(+),DhCpmFc(+)-(G₄S)₄-GDF15(H6D):DhCpmFc(−),DhCpmFc(+)-(G₄S)₄-GDF15(N3Q):DhCpmFc(−), DhCpmFc(+)-GDF15:DhCpmFc(−),DhCpmFc(+)-G₄-GDF15:DhCpmFc(−), DhCpmFc(+)-(G₄S)₂-GDF15:DhCpmFc(−),DhCpmFc(+)-(G₄Q)₄-GDF15:DhCpmFc(−),DhCpmFc(+)(L351C)-G₄-GDF15:DhCpmFc(−)(L351C) orDhCpmFc(+)(S354C)-G₄-GDF15:DhCpmFc(−)(Y349C).

In some embodiments, the fusion protein is not selected from the groupconsisting of Fc-(G₄S)₈-Fc-GS(G₄S)₄-GDF15, Fc-(G₄S)₃-Fc-GS(G₄S)₄-GDF15and Fc-(G₄S)₅-Fc-GS(G₄S)₄-GDF15.

In some embodiments, the fusion protein is not selected from the groupconsisting of DhCpmFc(+)(L351C)-G₄-GDF15:DhCpmFc(−)(L351C) andDhCpmFc(+)(S354C)-G₄-GDF15:DhCpmFc(−)(Y349C).

In some embodiments, the dimer is notDhCpmFc(−)-(G₄S)₄-GDF15:DhCpmFc(+). In some embodiments, the dimer isnot DhCpmFc(+)-(G₄S)₄-GDF15:DhCpmFc(−). In some embodiments, the dimeris not DhCpmFc(−)-(G₄S)₄-GDF15(H6D):DhCpmFc(+). In some embodiments, thedimer is not DhCpmFc(+)-(G₄S)₄-GDF15(H6D):DhCpmFc(−). In someembodiments, the dimer is not DhCpmFc(+)-(G₄S)₄-GDF15(N3Q):DhCpmFc(−).In some embodiments, the dimer is not DhCpmFc(+)-GDF15:DhCpmFc(−). Insome embodiments, the dimer is not DhCpmFc(+)-G₄-GDF15:DhCpmFc(−). Insome embodiments, the dimer is not DhCpmFc(+)-(G₄S)₂-GDF15:DhCpmFc(−).In some embodiments, the dimer is notDhCpmFc(+)-(G₄Q)₄-GDF15:DhCpmFc(−). In some embodiments, the dimer isnot DhCpmFc(+)(L351C)-G₄-GDF15:DhCpmFc(−)(L351C). In some embodiments,the dimer is not DhCpmFc(+)(S354C)-G₄-GDF15:DhCpmFc(−)(Y349C). In someembodiments, the dimer is not CpmFc(−)-(G₄S)₄-GDF15:CpmFc(+). In someembodiments, the dimer is not Fc-(G₄S)₈-Fc-GS(G₄S)₄-GDF15. In someembodiments, the dimer is not Fc-(G₄S)₃-Fc-GS(G₄S)₄-GDF15. In someembodiments, the dimer is not Fc-(G₄S)₅-Fc-GS(G₄S)₄-GDF15.

Also provided herein are fusion proteins comprising a GDF15 polypeptideor a GDF15 mutant polypeptide and a human serum albumin (HSA)polypeptide. In a further embodiment, the HSA polypeptide the sequenceof SEQ ID NO:110. In another embodiment, the GDF15 polypeptide or aGDF15 mutant polypeptide comprises a sequence selected from the groupconsisting of SEQ ID NOs:4, 8, 12, 25, 52 and 55. In another embodiment,the fusion protein further comprises a polypeptide linker, joining theGDF15 polypeptide or GDF15 mutant polypeptide to the HSA polypeptide. Inone embodiment, the polypeptide linker has a sequence selected from thegroup consisting of SEQ ID NOs:18, 30, 34, 40, 58, 61, 64, 69, 72, 75,78, 113, 116, 119, 122, 125, 128. In one embodiment, the fusion proteincomprises two or more HSA polypeptides. In one embodiment, the fusionprotein comprises a sequence selected from the group consisting of SEQID NOs: 115, 118, 121, 124, 127 and 130.

Also provided herein are dimers comprising (i) a first polypeptide chaincomprising a fusion protein comprising a first GDF15 region and a firstHSA polypeptide, and (ii) a second polypeptide chain comprising a secondHSA polypeptide. In some embodiments, the second polypeptide chainfurther comprises a second GDF15 region. In some embodiments, the dimeris a heterodimer (i.e., the first and second polypeptide chain havedifferent sequences). In some embodiments, the dimer is a homodimer(i.e., the first and second polypeptide chain have the same sequence).In some embodiments, the first polypeptide chain comprises a sequenceselected from the group consisting of SEQ ID NOs: 115, 118, 121, 124,127 and 130. In some embodiments, the second polypeptide chain comprisesa sequence selected from the group consisting of SEQ ID NOs: 115, 118,121, 124, 127 and 130. In one embodiment, the first and secondpolypeptide chains are non-covalently associated. In another embodiment,the first and second polypeptide chains are covalently associated. Inone embodiment, the second polypeptide chain comprises a second GDF15region and first and second polypeptide chains are covalently associatedvia disulfide bonds between their respective GDF15 regions. In anotherembodiment, the first and second polypeptide chains are associated byboth covalent and non-covalent interactions.

Also provided herein are dimers comprising (i) a first polypeptide chaincomprising a fusion protein comprising a first GDF15 region and an HSApolypeptide, and (ii) a second polypeptide chain comprising a secondGDF15 region. In some embodiments, the first polypeptide chain comprisesa sequence selected from the group consisting of SEQ ID NOs: 115, 118,121, 124, 127 and 130. In some embodiments, the second polypeptide chaincomprises a sequence selected from the group consisting of SEQ ID NOs:4, 8, 12, 25, 52 and 55. In one embodiment, the first and secondpolypeptide chains are non-covalently associated. In another embodiment,the first and second polypeptide chains are covalently associated. Inanother embodiment, the first and second polypeptide chains areassociated by both covalent and non-covalent interactions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphic depicting of a knob-hole construct comprising adimer of two DhknobFc-(G₄S)₄-GDF15:DhholeFc heterodimers.

FIG. 2 is a graphic depicting a DhMonoFc construct comprising a dimer oftwo DhMonoFc-(G₄S)₄-GDF15 fusion proteins.

FIG. 3 is a graphic depicting a HemiFc construct comprising a dimer oftwo GGGFc-(G₄S)₄-Fc-S(G₄S)₄-GDF15 fusion proteins.

FIG. 4 is a graphic depicting a charged pair (delHinge) constructcomprising a dimer of two DhCpmFc(−)-(G₄S)₂-GDF15:DhCpmFc(+)heterodimers.

FIG. 5 is a graphic depicting a charged pair (delHinge) cysteine clampconstruct comprising a dimer of twoDhCpmFc(−)(L351C)-(G₄S)₂-GDF15:DhCpmFc(−)(L351C) heterodimers.

FIG. 6 is a graphic depicting an HSA construct comprising a dimer of twoHSA-(G₄S)₄-GDF15 fusion proteins.

FIG. 7 is a plot showing the effect on food intake (g food/g body weight(BW) of ob/ob mice as a function of dose (log [g construct/kg BW]) usinga dimer of the DhknobFc-G₄-GDF15:DhholeFc heterodimer.

FIG. 8 is a plot showing the effect on food intake (g food/g bodyweight) of ob/ob mice as a function of dose (log [g construct/kg BW])using a dimer of the Fc-(G₄S)₄-GDF15 fusion protein.

FIG. 9 is a plot showing the effect on food intake (g food/g bodyweight) of ob/ob mice as a function of dose (log [g construct/kg BW])using a dimer of the Fc-(G₄S)₄-GDF15(H6D) fusion protein.

FIG. 10 is a plot showing the effect on food intake (g food/g bodyweight) of ob/ob mice as a function of dose (log [g construct/kg BW])using a dimer of the Fc-(G₄S)₄-Fc-(G₄S)₄-GDF15 fusion protein.

FIG. 11 is a plot showing the effect on food intake (g food/g bodyweight) of ob/ob mice as a function of dose (log [g construct/kg BW])using a dimer of the DhFc-(G₄S)₅-DhFc-(G₄S)₄-GDF15 fusion protein.

FIG. 12 is a plot showing the effect on food intake (g food/g bodyweight) of ob/ob mice as a function of dose (log [g construct/kg BW])using a dimer of the DhCpmFc(+)-(1K)-GDF15:DhCpmFc(−) heterodimer.

FIG. 13 is a plot showing the effect on food intake (g food/g bodyweight) of ob/ob mice as a function of dose (log [g construct/kg BW])using a dimer of the DhCpmFc(−)-GDF15:DhCpmFc(+) heterodimer.

FIG. 14 is a plot showing the effect on food intake (g food/g bodyweight) of ob/ob mice as a function of dose (log [g construct/kg BW])using a dimer of the DhCpmFc(−)-GDF15(N3D):DhCpmFc(+) heterodimer.

FIG. 15 is a plot showing the effect on food intake (g food/g bodyweight) of ob/ob mice as a function of dose (log [g construct/kg BW])using a dimer of the DhCpmFc(−)-GDF15(Ndel3):DhCpmFc(+) heterodimer.

FIG. 16 is a plot showing the effect on food intake (g food/g bodyweight) of ob/ob mice as a function of dose (log [g construct/kg BW])using a dimer of the DhCpmFc(−)-G₄-GDF15(N3D):DhCpmFc(+) heterodimer.

FIG. 17 is a plot showing the effect on food intake (g food/g bodyweight) of ob/ob mice as a function of dose (log [g construct/kg BW])using a dimer of the DhCpmFc(−)-G₄S-GDF15:DhCpmFc(+) heterodimer.

FIG. 18 is a plot showing the effect on food intake (g food/g bodyweight) of ob/ob mice as a function of dose (log [g construct/kg BW])using a dimer of the DhCpmFc(−)-(G₄S)₂-GDF15:DhCpmFc(+) heterodimer.

FIG. 19 is a plot showing the effect on food intake (g food/g bodyweight) of ob/ob mice as a function of dose (log [g construct/kg BW])using a dimer of the DhCpmFc(−)-(G₄S)₂-GDF15(N3D):DhCpmFc(+)heterodimer.

FIG. 20 is a plot showing the effect on food intake (g food/g bodyweight) of ob/ob mice as a function of dose (log [g construct/kg BW])using a dimer of the DhCpmFc(−)-(G₄Q)₂-GDF15(N3D):DhCpmFc(+)heterodimer.

FIG. 21 is a plot showing the effect on food intake (g food/g bodyweight) of ob/ob mice as a function of dose (log [g construct/kg BW])using a dimer of the DhCpmFc(−)-G₄P-GDF15:DhCpmFc(+) heterodimer.

FIG. 22 is a plot showing the effect on food intake (g food/g bodyweight) of ob/ob mice as a function of dose (log [g construct/kg BW])using a dimer of the DhCpmFc(−)-(G₄P)₂-GDF15:DhCpmFc(+) heterodimer.

FIG. 23 is a plot showing the effect on food intake (g food/g bodyweight) of ob/ob mice as a function of dose (log [g construct/kg BW])using a dimer of the DhCpmFc(−)-G₄Q-GDF15:DhCpmFc(+) heterodimer.

FIG. 24 is a plot showing the effect on food intake (g food/g bodyweight) of ob/ob mice as a function of dose (log [g construct/kg BW])using a dimer of the DhCpmFc(−)-(G₄Q)₂-GDF15(N3D):DhCpmFc(+)heterodimer.

FIG. 25 is a plot showing the effect on food intake (g food/g bodyweight) of ob/ob mice as a function of dose (log [g construct/kg BW])using a dimer of the DhCpmFc(−)-(G₄Q)₂-GDF15(Ndel3):DhCpmFc(+)heterodimer.

FIG. 26 is a plot showing the effect on food intake (g food/g bodyweight) of ob/ob mice as a function of dose (log [g construct/kg BW])using a dimer of the DhCpmFc(+)(Y349C)-GDF15(N3D):DhCpmFc(−)(S354C)heterodimer.

FIG. 27 is a plot showing the effect on food intake (g food/g bodyweight) of ob/ob mice as a function of dose (log [g construct/kg BW])using a dimer of the DhCpmFc(−)(Y349C)-GDF15:DhCpmFc(+)(S354C)heterodimer.

FIG. 28 is a plot showing the effect on food intake (g food/g bodyweight) of ob/ob mice as a function of dose (log [g construct/kg BW])using a dimer of the DhCpmFc(−)(Y349C)-GDF15(N3D):DhCpmFc(+)(S354C)heterodimer.

FIG. 29 is a plot showing the effect on food intake (g food/g bodyweight) of ob/ob mice as a function of dose (log [g construct/kg BW])using a dimer of the DhCpmFc(−)(Y349C)-GDF15(Ndel3):DhCpmFc(+)(S354C)heterodimer.

FIG. 30 is a plot showing the effect on food intake (g food/g bodyweight) of ob/ob mice as a function of dose (log [g construct/kg BW])using a dimer of the DhCpmFc(−)(Y349C)-G₄-GDF15(N3D):DhCpmFc(+)(S354C)heterodimer.

FIG. 31 is a plot showing the effect on food intake (g food/g bodyweight) of ob/ob mice as a function of dose (log [g construct/kg BW])using a dimer of theDhCpmFc(−)(Y349C)-(G₄S)₂-GDF15(N3D):DhCpmFc(+)(S354C) heterodimer.

FIG. 32 is a plot showing the effect on food intake (g food/g bodyweight) of ob/ob mice as a function of dose (log [g construct/kg BW])using a dimer of theDhCpmFc(−)(Y349C)-(G₄Q)₂-GDF15(N3D):DhCpmFc(+)(S354C) heterodimer.

FIG. 33 is a plot showing the effect on food intake (g food/g bodyweight) of ob/ob mice as a function of dose (log [g construct/kg BW])using a dimer of the DhCpmFc(−)(L351C)-(G₄S)₂-GDF15:DhCpmFc(+)(L351C)heterodimer.

FIG. 34 is a plot showing the effect on food intake (g food/g bodyweight) of ob/ob mice as a function of dose (log [g construct/kg BW])using a dimer of the HSA-GSAAQAAQQGS-GDF 15 fusion protein.

FIG. 35 is a plot showing the effect on food intake (g food/g bodyweight) of ob/ob mice as a function of dose (log [g construct/kg BW])using a dimer of the HSA-GSPAPAPGS-GDF 15 fusion protein.

FIG. 36 is a plot showing the effect on food intake (g food/g bodyweight) of ob/ob mice as a function of dose (log [g construct/kg BW])using a dimer of the HSA-GS(AAQAAQQ)₂GS-GDF15 fusion protein.

FIG. 37 is a plot showing the effect on food intake (g food/g bodyweight) of ob/ob mice as a function of dose (log [g construct/kg BW])using a dimer of the HSA-GS(PAPAP)₂GS-GDF15 fusion protein.

FIG. 38 is a plot showing the effect on food intake (g food/g bodyweight) of ob/ob mice as a function of dose (log [g construct/kg BW])using a dimer of the HSA-GDF15 fusion protein.

FIG. 39 is a plot showing the effect on food intake (g food/g bodyweight) of ob/ob mice as a function of dose (log [g construct/kg BW])using a dimer of the HSA-GGNAEAAAKEAAAKEAAAKAGG-GDF 15 fusion protein.

FIG. 40 is a plot showing the effect on food intake (g food/g bodyweight) of ob/ob mice as a function of dose (log [g construct/kg BW])using a dimer of the HSA-(G₄S)₆-GDF15 fusion protein.

FIG. 41 is a plot showing the effect on food intake (g food/g bodyweight) of ob/ob mice as a function of dose (log [g construct/kg BW])using a dimer of the DhCpmFc(−)(N297G)-GDF15(Ndel3):DhCpmFc(+)(N297G)heterodimer.

FIG. 42 is a plot showing the effect on food intake (g food/g bodyweight) of ob/ob mice as a function of dose (log [g construct/kg BW])using a dimer of the DhCpmFc(−)(N297G)-GDF15(N3D):DhCpmFc(+)(N297G)heterodimer.

FIG. 43 is a plot showing the effect on food intake (g food/g bodyweight) of ob/ob mice as a function of dose (log [g construct/kg BW])using a dimer of theDhCpmFc(−)(N297G)(Y349C)-GDF15(Ndel3):DhCpmFc(+)(N297G)(S354C)heterodimer.

FIG. 44 is a plot showing the effect on food intake (g food/g bodyweight) of ob/ob mice as a function of dose (log [g construct/kg BW])using a dimer of theDhCpmFc(−)(N297G)(Y349C)-GDF15(N3D):DhCpmFc(+)(N297G)(S354C)heterodimer.

FIG. 45 is a plot showing the effect on food intake (g food/g bodyweight) of ob/ob mice as a function of dose (log [g construct/kg BW])using a dimer of theDhCpmFc(−)(N297G)(L351C)-GDF15(Ndel3):DhCpmFc(+)(N297G)(L351C)heterodimer.

FIG. 46 is a plot showing the effect on food intake (g food/g bodyweight) of ob/ob mice as a function of dose (log [g construct/kg BW])using a dimer of theDhCpmFc(−)(N297G)(L351C)-GDF15(N3D):DhCpmFc(+)(N297G)(L351C)heterodimer.

FIG. 47 is a plot showing the effect on food intake (g food/g bodyweight) of ob/ob mice as a function of dose (log [g construct/kg BW])using a dimer of theDhCpmFc(−)(N297G)(A287C)-GDF15(Ndel3):DhCpmFc(+)(N297G)(L306C)heterodimer.

FIG. 48 is a plot showing the effect on food intake (g food/g bodyweight) of ob/ob mice as a function of dose (log [g construct/kg BW])using a dimer of the CpmFc(−)(N297G)-GDF15(Ndel3):CpmFc(+)(N297G)heterodimer.

FIG. 49 is a plot showing the effect on food intake (g food/g bodyweight) of ob/ob mice as a function of dose (log [g construct/kg BW])using a dimer of the Dh3CpmFc(−)-GDF15(Ndel3):Dh3CpmFc(N297G)heterodimer.

FIG. 50 is a plot showing the effect on food intake (g food/g bodyweight) of ob/ob mice as a function of dose (log [g construct/kg BW])using a dimer of the Dh3CpmFc(−)-GDF15(N3D):Dh3CpmFc(+)(N297G)heterodimer.

FIG. 51 is a plot showing the effect on food intake (g food/g bodyweight) of ob/ob mice as a function of dose (log [g construct/kg BW])using a dimer of theDh3CpmFc(−)(Y349C)-GDF15(Ndel3):Dh3CpmFc(+)(N297G)(S354C) heterodimer.

FIG. 52 is a plot showing the effect on food intake (g food/g bodyweight) of ob/ob mice as a function of dose (log [g construct/kg BW])using a dimer of theDh3CpmFc(−)(Y349C)-GDF15(N3D):Dh3CpmFc(−)(N297G)(S354C) heterodimer.

FIG. 53 is a plot showing the effect on food intake (g food/g bodyweight) of ob/ob mice as a function of dose (log [g construct/kg BW])using a dimer of the DhMonoFc(N297G)-GDF15 fusion protein.

FIG. 54 is a plot of body weight (g) as a function of time (days post1^(st) injection) for vehicle, 0.1 nmol, 1 nmol and 10 nmol of a dimerof the Dh3CpmFc(−)-GDF15(N3D):Dh3CpmFc(+) heterodimer, and 0.1 nmol, 1nmol and 10 nmol of a dimer of the Dh3ComFc(−)-GDF15(Ndel3) heterodimer.

FIG. 55 is a bar graph of area under the curve (AUC) for a glucosetolerance test at week 2 of treatment with (a) vehicle (b) 10 mmol of adimer of the Dh3CpmFc(−)-GDF15(N3D):Dh3CpmFc(+) heterodimer (c) 1 mmolof a dimer of the Dh3CpmFc(−)-GDF15(N3D):Dh3CpmFc(+) heterodimer, (d)0.1 mmol of a dimer of the Dh3CpmFc(−)-GDF15(N3D):Dh3CpmFc(+)heterodimer, (e) 10 mmol of a dimer of theDh3CpmFc(−)-GDF15(Ndel3):Dh3CpmFc(+) heterodimer (g) 1 mmol of a dimerof the Dh3CpmFc(−)-GDF15(Ndel3):Dh3CpmFc(+) heterodimer, or (g) 0.1 mmolof a dimer of the Dh3CpmFc(−)-GDF15(Ndel):Dh3CpmFc(+) heterodimer.

FIG. 56 is a bar graph of insulin (ng/mL) (fed) at week 3 of treatmentwith (a) vehicle (b) 10 mmol of a dimer of theDh3CpmFc(−)-GDF15(N3D):Dh3CpmFc(+) heterodimer (c) 1 mmol of a dimer ofthe Dh3CpmFc(−)-GDF15(N3D):Dh3CpmFc(+) heterodimer, (d) 0.1 mmol of adimer of the Dh3CpmFc(−)-GDF15(N3D):Dh3CpmFc(+) heterodimer, (e) 10 mmolof a dimer of the Dh3CpmFc(−)-GDF15(Ndel3):Dh3CpmFc(+) heterodimer (g) 1mmol of a dimer of the Dh3CpmFc(−)-GDF15(Ndel3):Dh3CpmFc(+) heterodimer,or (g) 0.1 mmol of a dimer of the Dh3CpmFc(−)-GDF15(Ndel):Dh3CpmFc(+)heterodimer.

FIG. 57 is a bar graph of triglycerides (mg/mL) (fed) at week 3 oftreatment with (a) vehicle (b) 10 mmol of a dimer of theDh3CpmFc(−)-GDF15(N3D):Dh3CpmFc(+) heterodimer (c) 1 mmol of a dimer ofthe Dh3CpmFc(−)-GDF15(N3D):Dh3CpmFc(+) heterodimer, (d) 0.1 mmol of adimer of the Dh3CpmFc(−)-GDF15(N3D):Dh3CpmFc(+) heterodimer, (e) 10 mmolof a dimer of the Dh3CpmFc(−)-GDF15(Ndel3):Dh3CpmFc(+) heterodimer (g) 1mmol of a dimer of the Dh3CpmFc(−)-GDF15(Ndel3):Dh3CpmFc(+) heterodimer,or (g) 0.1 mmol of a dimer of the Dh3CpmFc(−)-GDF15(Ndel):Dh3CpmFc(+)heterodimer.

FIG. 58 is a bar graph of cholesterol (mg/mL) (fed) at week 3 oftreatment with (a) vehicle (b) 10 mmol of a dimer of theDh3CpmFc(−)-GDF15(N3D):Dh3CpmFc(+) heterodimer (c) 1 mmol of a dimer ofthe Dh3CpmFc(−)-GDF15(N3D):Dh3CpmFc(+) heterodimer, (d) 0.1 mmol of adimer of the Dh3CpmFc(−)-GDF15(N3D):Dh3CpmFc(+) heterodimer, (e) 10 mmolof a dimer of the Dh3CpmFc(−)-GDF15(Ndel3):Dh3CpmFc(+) heterodimer (g) 1mmol of a dimer of the Dh3CpmFc(−)-GDF15(Ndel3):Dh3CpmFc(+) heterodimer,or (g) 0.1 mmol of a dimer of the Dh3CpmFc(−)-GDF15(Ndel):Dh3CpmFc(+)heterodimer.

FIG. 59 is a bar graph of area under the curve (AUC) for a glucosetolerance test at week 5 of treatment with (a) vehicle (b) 10 mmol of adimer of the Dh3CpmFc(−)-GDF15(N3D):Dh3CpmFc(+) heterodimer (c) 1 mmolof a dimer of the Dh3CpmFc(−)-GDF15(N3D):Dh3CpmFc(+) heterodimer, (d)0.1 mmol of a dimer of the Dh3CpmFc(−)-GDF15(N3D):Dh3CpmFc(+)heterodimer, (e) 10 mmol of a dimer of theDh3CpmFc(−)-GDF15(Ndel3):Dh3CpmFc(+) heterodimer (g) 1 mmol of a dimerof the Dh3CpmFc(−)-GDF15(Ndel3):Dh3CpmFc(+) heterodimer, or (g) 0.1 mmolof a dimer of the Dh3CpmFc(−)-GDF15(Ndel):Dh3CpmFc(+) heterodimer.

FIG. 60 is a bar graph showing SPR Binding (RU) with respect to FcγRI,FcγRIIIA and FcγRIIA for (a) a dimer ofDhCpmFc(−)-GDF15(Ndel3):DhCpmFc(+), (b) a dimer ofDhCpmFc(−)(Y349C)-GDF15(Ndel3):DhCpmFc(+)(S354C); (c) a dimer ofDh3CpmFc(−)-GDF15(Ndel3); (d) a dimer ofDh3CpmFc(−)(Y349C)-GDF15(Ndel3)-Dh3CpmFc(+)(S354C); (e) a dimer ofDh3CpmFc(−)-GDF15(N3D); and (f) a dimer ofDh3CpmFc(−)(Y349C)-GDF15(N3D):Dh3CpmFc(+)(S354C) (from left to rightwith respect to each receptor).

FIG. 61 is a bar graph showing DSC first Tm (° C.) with respect to (a)(for pair of bars captioned “N3D”), a dimer ofDhCpmFc(−)-GDF15(N3D):DhCpmFc(+) and a dimer ofDh3CpmFc(−)-GDF15(N3D):DhCpmFc(+); (b) (for the pair of bars captioned“Ndel3”), a dimer of DhCpmFc(−)-GDF15(Ndel3):DhCpmFc(+) and a dimer ofDh3CpmFc(−)-GDF15(Ndel3):DhCpmFc(+); (c) (for the pair of bars captioned“N3D+CC”), a dimer of DhCpmFc(−)(Y349C)-GDF15(N3D):DhCpmFc(+)(S354C) anda dimer of Dh3CpmFc(−) (Y349C)-GDF15(N3D):DhCpmFc(+)(S354C); and (d)(for the pair of bars captioned “Ndel3+CC”), a dimer ofDhCpmFc(−)(Y349C)-GDF15(Ndel3):DhCpmFc(+)(S354C) and a dimer ofDh3CpmFc(−)(Y349C)-GDF15(Ndel3):DhCpmFc(+)(S354C).

DETAILED DESCRIPTION OF THE INVENTION

The instant disclosure provides fusion proteins comprising a GDF15polypeptide or a GDF15 mutant polypeptides and constructs comprisingsuch fusion proteins. Also provided is the generation and uses of thedisclosed molecules, for example in treating a metabolic disorder, suchas Type 2 diabetes, elevated glucose levels, elevated insulin levels,dyslipidemia or obesity. GDF15 polypeptides, GDF15 mutant polypeptides,and certain polypeptide constructs comprising GDF15 polypeptides andGDF15 mutant polypeptides, are described in co-owned PCT/US2012/032415,filed Apr. 5, 2012, and PCT/2013/023465, filed Jan. 28, 2013, both ofwhich are expressly incorporated by reference herein for any purpose.

Recombinant polypeptide and nucleic acid methods used herein, includingin the Examples, are generally those set forth in Sambrook et al.,Molecular Cloning: A Laboratory Manual (Cold Spring Harbor LaboratoryPress, 1989) or Current Protocols in Molecular Biology (Ausubel et al.,eds., Green Publishers Inc. and Wiley and Sons 1994).

I. General Definitions

Following convention, as used herein “a” and “an” mean “one or more”unless specifically indicated otherwise.

As used herein, the terms “amino acid” and “residue” are interchangeableand, when used in the context of a peptide or polypeptide, refer to bothnaturally-occurring and synthetic amino acids, as well as amino acidanalogs, amino acid mimetics and non-naturally-occurring amino acidsthat are chemically similar to the naturally-occurring amino acids.

The terms “naturally-occurring amino acid” and “naturally encoded aminoacid” are used interchangeably and refer to an amino acid that isencoded by the genetic code, as well as those amino acids that areencoded by the genetic code that are modified after synthesis, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine.

An “amino acid analog” is a compound that has the same basic chemicalstructure as a naturally-occurring amino acid, i.e., an α carbon that isbound to a hydrogen, a carboxyl group, an amino group, and an R group,e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs can have modified R groups (e.g., norleucine) ormodified peptide backbones, but will retain the same basic chemicalstructure as a naturally-occurring amino acid.

An “amino acid mimetic” is a chemical compound that has a structure thatis different from the general chemical structure of an amino acid, butthat functions in a manner similar to a naturally-occurring amino acid.Examples include a methacryloyl or acryloyl derivative of an amide, β-,γ-, δ-imino acids (such as piperidine-4-carboxylic acid) and the like.

The terms “non-naturally-occurring amino acid” and “non-naturallyencoded amino acid” are used interchangeably and refer to a compoundthat has the same basic chemical structure as a naturally-occurringamino acid, but is not incorporated into a growing polypeptide chain bythe translation complex. “Non-naturally-occurring amino acid” alsoincludes, but is not limited to, amino acids that occur by modification(e.g., posttranslational modifications) of a naturally encoded aminoacid (including but not limited to, the 20 common amino acids) but arenot themselves naturally incorporated into a growing polypeptide chainby the translation complex. A non-limiting lists of examples ofnon-naturally-occurring amino acids that can be inserted into apolypeptide sequence or substituted for a wild-type residue inpolypeptide sequence include β-amino acids, homoamino acids, cyclicamino acids and amino acids with derivatized side chains. Examplesinclude (in the L-form or D-form; abbreviated as in parentheses):citrulline (Cit), homocitrulline (hCit), Nα-methylcitrulline (NMeCit),Nα-methylhomocitrulline (Nα-MeHoCit), ornithine (Orn),Nα-Methylornithine (Nα-MeOrn or NMeOrn), sarcosine (Sar), homolysine(hLys or hK), homoarginine (hArg or hR), homoglutamine (hQ),Nα-methylarginine (NMeR), Nα-methylleucine (Nα-MeL or NMeL),N-methylhomolysine (NMeHoK), Nα-methylglutamine (NMeQ), norleucine(Nle), norvaline (Nva), 1,2,3,4-tetrahydroisoquinoline (Tic),Octahydroindole-2-carboxylic acid (Oic), 3-(1-naphthyl)alanine (1-Nal),3-(2-naphthyl)alanine (2-Nal), 1,2,3,4-tetrahydroisoquinoline (Tic),2-indanylglycine (IgI), para-iodophenylalanine (pI-Phe),para-aminophenylalanine (4AmP or 4-Amino-Phe), 4-guanidino phenylalanine(Guf), glycyllysine (abbreviated “K(Nε-glycyl)” or “K(glycyl)” or“K(gly)”), nitrophenylalanine (nitrophe), aminophenylalanine (aminopheor Amino-Phe), benzylphenylalanine (benzylphe), γ-carboxyglutamic acid(γ-carboxyglu), hydroxyproline (hydroxypro), p-carboxyl-phenylalanine(Cpa), α-aminoadipic acid (Aad), Nα-methyl valine (NMeVal), N-α-methylleucine (NMeLeu), Nα-methylnorleucine (NMeNle), cyclopentylglycine(Cpg), cyclohexylglycine (Chg), acetylarginine (acetylarg), α,β-diaminopropionoic acid (Dpr), α, γ-diaminobutyric acid (Dab),diaminopropionic acid (Dap), cyclohexylalanine (Cha),4-methyl-phenylalanine (MePhe), β, β-diphenyl-alanine (BiPhA),aminobutyric acid (Abu), 4-phenyl-phenylalanine (or biphenylalanine;4Bip), α-amino-isobutyric acid (Aib), beta-alanine, beta-aminopropionicacid, piperidinic acid, aminocaprioic acid, aminoheptanoic acid,aminopimelic acid, desmosine, diaminopimelic acid, N-ethylglycine,N-ethylaspargine, hydroxylysine, allo-hydroxylysine, isodesmosine,allo-isoleucine, N-methylglycine, N-methylisoleucine, N-methylvaline,4-hydroxyproline (Hyp), γ-carboxyglutamate, ε-N,N,N-trimethyllysine,ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine,3-methylhistidine, 5-hydroxylysine, ω-methylarginine, 4-Amino-O-PhthalicAcid (4APA), N-acetylglucosaminyl-L-serine,N-acetylglucosylaminyl-L-threonine, O-phosphotyrosine and other similaramino acids, and derivatized forms of any of those specifically listed.

Also included in the definition of “non-naturally-occurring amino acid”is any amino acid comprising the structure

wherein the R group is any substituent other than the one used in thetwenty natural amino acids.

In some embodiments, the non-naturally encoded amino acid comprises acarbonyl group. In some embodiments, the non-naturally encoded aminoacid has the structure:

wherein n is 0-10; R₁ is an alkyl, aryl, substituted alkyl, orsubstituted aryl; R₂ is H, an alkyl, aryl, substituted alkyl, andsubstituted aryl; and R₃ is H, an amino acid, a polypeptide, or an aminoterminus modification group, and R₄ is H, an amino acid, a polypeptide,or a carboxy terminus modification group.

In some embodiments, the non-naturally encoded amino acid comprises anaminooxy group. In some embodiments, the non-naturally encoded aminoacid comprises a hydrazide group. In some embodiments, the non-naturallyencoded amino acid comprises a hydrazine group. In some embodiments, thenon-naturally encoded amino acid residue comprises a semicarbazidegroup.

In some embodiments, the non-naturally encoded amino acid residuecomprises an azide group. In some embodiments, the non-naturally encodedamino acid has the structure:

wherein n is 0-10; R₁ is an alkyl, aryl, substituted alkyl, substitutedaryl or not present; X is O, N, S or not present; m is 0-10; R₂ is H, anamino acid, a polypeptide, or an amino terminus modification group, andR₃ is H, an amino acid, a polypeptide, or a carboxy terminusmodification group.

In some embodiments, the non-naturally encoded amino acid comprises analkyne group. In some embodiments, the non-naturally encoded amino acidhas the structure:

wherein n is 0-10; R₁ is an alkyl, aryl, substituted alkyl, orsubstituted aryl; X is O, N, S or not present; m is 0-10, R₂ is H, anamino acid, a polypeptide, or an amino terminus modification group, andR₃ is H, an amino acid, a polypeptide, or a carboxy terminusmodification group.

The term “substituted” means that a hydrogen atom on a molecule or groupis replaced with a group or atom, which is referred to as a substituent.Typical substituents include: halogen, C₁₋₈alkyl, hydroxyl, C₁₋₈alkoxy,—NR^(x)R^(x), nitro, cyano, halo or perhaloC₁₋₈alkyl, C₂₋₈alkenyl,C₂₋₈alkynyl, —SR^(x), —S(═O)₂R^(x), —C(═O)OR^(x), —C(═O)R^(x), whereineach R^(x) is independently hydrogen or C₁-C₈ alkyl. It is noted thatwhen the substituent is —NR^(x)R^(x), the R^(x) groups may be joinedtogether with the nitrogen atom to form a ring.

The term “alkyl” means a straight or branched chain hydrocarbon.Representative examples of alkyl groups include methyl, ethyl, propyl,isopropyl, butyl, isobutyl, tert-butyl, sec-butyl, pentyl and hexyl.Typical alkyl groups are alkyl groups having from 1 to 8 carbon atoms,which groups are commonly represented as C₁₋₈alkyl.

The term “alkoxy” means an alkyl group bonded to an oxygen atom.Representative examples of alkoxy groups include methoxy, ethoxy,tert-butoxy, propoxy and isobutoxy. Common alkoxy groups are C₁₋₈alkoxy.

The term “halogen” or “halo” means chlorine, fluorine, bromine oriodine.

The term “alkenyl” means a branched or straight chain hydrocarbon havingone or more carbon-carbon double bonds. Representative examples alkenylgroups include ethenyl, propenyl, allyl, butenyl and 4-methylbutenyl.Common alkenyl groups are C₂₋₈alkenyl.

The term “alkynyl” means a branched or straight chain hydrocarbon havingone or more carbon-carbon triple bonds. Representative examples ofalkynyl groups include ethynyl, propynyl (propargyl) and butynyl. Commonalkynyl groups are C₂₋₈ alkynyl.

The term “cycloalkyl” means a cyclic, nonaromatic hydrocarbon. Examplesof cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl and cycloheptyl. A cycloalkyl group can contain one or moredouble bond. Examples of cycloalkyl groups that contain double bondsinclude cyclopentenyl, cyclohexenyl, cyclohexadienyl andcyclobutadienyl. Common cycloalkyl groups are C₃₋₈ cycloalkyl groups.

The term “perfluoroalkyl” means an alkyl group in which all of thehydrogen atoms have been replaced with fluorine atoms. Commonperfluoroalkyl groups are C₁₋₈perfluoroalkyl. An example of a commonperfluoroalkyl group is —CF₃.

The term “acyl” means a group derived from an organic acid by removal ofthe hydroxy group (—OH). For example, the acyl group CH₃C(═O)— is formedby the removal of the hydroxy group from CH₃C(═O)OH.

The term “aryl” means a cyclic, aromatic hydrocarbon. Examples of arylgroups include phenyl and naphthyl. Common aryl groups are six tothirteen membered rings.

The term “heteroatom” as used herein means an oxygen, nitrogen or sulfuratom.

The term “heteroaryl” means a cyclic, aromatic hydrocarbon in which oneor more carbon atoms of an aryl group have been replaced with aheteroatom. If the heteroaryl group contains more than one heteroatom,the heteroatoms may be the same or different. Examples of heteroarylgroups include pyridyl, pyrimidinyl, imidazolyl, thienyl, furyl,pyrazinyl, pyrrolyl, indolyl, triazolyl, pyridazinyl, indazolyl,purinyl, quinolizinyl, isoquinolyl, quinolyl, naphthyridinyl,quinoxalinyl, isothiazolyl and benzo[b]thienyl. Common heteroaryl groupsare five to thirteen membered rings that contain from 1 to 4heteroatoms. Heteroaryl groups that are five and six membered rings thatcontain 1 to 3 heteroatoms are particularly common.

The term “heterocycloalkyl” means a cycloalkyl group in which one ormore of the carbon atoms has been replaced with a heteroatom. If theheterocycloalkyl group contains more than one heteroatom, theheteroatoms may be the same or different. Examples of heterocycloalkylgroups include tetrahydrofuryl, morpholinyl, piperazinyl, piperidinyland pyrrolidinyl. It is also possible for the heterocycloalkyl group tohave one or more double bonds, but is not aromatic. Examples ofheterocycloalkyl groups containing double bonds include dihydrofuran.Common heterocycloalkyl groups are three to ten membered ringscontaining from 1 to 4 heteroatoms. Heterocycloalkyl groups that arefive and six membered rings that contain 1 to 2 heteroatoms areparticularly common.

It is also noted that the cyclic ring groups, i.e., aryl, heteroaryl,cycloalkyl, and heterocycloalkyl, can comprise more than one ring. Forexample, the naphthyl group is a fused bicyclic ring system. It is alsointended that the present invention include ring groups that havebridging atoms, or ring groups that have a spiro orientation.

Representative examples of five to six membered aromatic rings,optionally having one or two heteroatoms, are phenyl, furyl, thienyl,pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl,isothiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, and pyrazinyl.

Representative examples of partially saturated, fully saturated or fullyunsaturated five to eight membered rings, optionally having one to threeheteroatoms, are cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl andphenyl. Further exemplary five membered rings are furyl, thienyl,pyrrolyl, 2-pyrrolinyl, 3-pyrrolinyl, pyrrolidinyl, 1,3-dioxolanyl,oxazolyl, thiazolyl, imidazolyl, 2H-imidazolyl, 2 imidazolinyl,imidazolidinyl, pyrazolyl, 2-pyrazolinyl, pyrazolidinyl, isoxazolyl,isothiazolyl, 1,2-dithiolyl, 1,3-dithiolyl, 3H-1,2-oxathiolyl,1,2,3-oxadizaolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl,1,3,4oxadiazolyl, 1,2,3 thazolyl, 1,2,4 trizaolyl, 1,3,4-thiadiazolyl,3H-1,2,3-dioxazolyl, 1,2,4-dioxazolyl, 1,3,2-dioxazolyl,1,3,4-dioxazolyl, 5H-1,2,5-oxathiazolyl, and 1,3-oxathiolyl.

Further exemplary six membered rings are 2H-pyranyl, 4H-pyranyl,pyridinyl, piperidinyl, 1,4-dioxanyl, morpholinyl, 1,4 dithianyl,thiomorpholinyl, pyndazinyl, pyrimidinyl, pyrazinyl, piperazinyl, 1,3,5triazinyl, 1,2,4 triazinyl, 4H-1,2-oxazinyl, 2H-1,3-oxazinyl,6H-1,3-oxazinyl, 6H-1,2-oxazinyl, 1,4-oxazinyl, 2H-1,2-oxazinyl,4H-1,4-oxazinyl, 1,2,5-oxathiazinyl, 1,4-oxazinyl, o-isoxazinyl,p-isoxazinyl, 1,2,5-oxathiazinyl, 1,2,6-(3 oxathiazinyl, and1,4,2-oxadiazinyl.

Further exemplary seven membered rings are azepinyl, oxepinyl, thiepinyland 1,2,4-triazepinyl.

Further exemplary eight membered rings are cyclooctyl, cyclooctenyl andcyclooctadienyl.

Exemplary bicyclic rings consisting of two fused partially saturated,fully saturated or fully unsaturated five and/or six membered rings,optionally having one to four heteroatoms, are indolizinyl, indolyl,isoindolyl, indolinyl, cyclopenta(b)pyridinyl, pyrano(3,4-b)pyrrolyl,benzofuryl, isobenzofuryl, benzo(b)thienyl, benzo(c)thienyl,1H-indazolyl, indoxazinyl, benzoxazolyl, anthranilyl, benzimidazolyl,benzthiazolyl, purinyl, quinolinyl, isoquinolinyl, cinnolinyl,phthalazinyl, quinazolinyl, quinoxalinyl, 1,8-naphthyridinyl,pteridinyl, indenyl, isoindenyl, naphthyl, tetralinyl, decalinyl,2H-1-benzopyranyl, pyrido(3,4-b)pyridinyl, pyrido(3,2-b)pyridinyl,pyrido(4,3-b)-pyridinyl, 2H-1,3-benzoxazinyl, 2H-1,4-benzoxazinyl,1H-2,3-benzoxazinyl, 4H-3,1-benzoxazinyl, 2H-1,2-benzoxazinyl and4H-1,4-benzoxazinyl.

A cyclic ring group may be bonded to another group in more than one way.If no particular bonding arrangement is specified, then all possiblearrangements are intended. For example, the term “pyridyl” includes 2-,3-, or 4-pyridyl, and the term “thienyl” includes 2-, or 3-thienyl.

The term “isolated nucleic acid molecule” refers to a single ordouble-stranded polymer of deoxyribonucleotide or ribonucleotide basesread from the 5′ to the 3′ end (e.g., a GDF15 nucleic acid sequenceprovided herein), or an analog thereof, that has been separated from atleast about 50 percent of polypeptides, peptides, lipids, carbohydrates,polynucleotides or other materials with which the nucleic acid isnaturally found when total nucleic acid is isolated from the sourcecells. Preferably, an isolated nucleic acid molecule is substantiallyfree from any other contaminating nucleic acid molecules or othermolecules that are found in the natural environment of the nucleic acidthat would interfere with its use in polypeptide production or itstherapeutic, diagnostic, prophylactic or research use.

The term “isolated polypeptide” refers to a polypeptide (e.g., a GDF15polypeptide or GDF15 mutant polypeptide provided herein) that has beenseparated from at least about 50 percent of polypeptides, peptides,lipids, carbohydrates, polynucleotides, or other materials with whichthe polypeptide is naturally found when isolated from a source cell.Preferably, the isolated polypeptide is substantially free from anyother contaminating polypeptides or other contaminants that are found inits natural environment that would interfere with its therapeutic,diagnostic, prophylactic or research use.

The term “encoding” refers to a polynucleotide sequence encoding one ormore amino acids. The term does not require a start or stop codon. Anamino acid sequence can be encoded in any one of the different readingframes provided by a polynucleotide sequence.

The terms “identical” and percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same. “Percent identity” meansthe percent of identical residues between the amino acids or nucleotidesin the compared molecules and is calculated based on the size of thesmallest of the molecules being compared. For these calculations, gapsin alignments (if any) can be addressed by a particular mathematicalmodel or computer program (i.e., an “algorithm”). Methods that can beused to calculate the identity of the aligned nucleic acids orpolypeptides include those described in Computational Molecular Biology,(Lesk, A. M., ed.), (1988) New York: Oxford University Press;Biocomputing Informatics and Genome Projects, (Smith, D. W., ed.), 1993,New York: Academic Press; Computer Analysis of Sequence Data, Part I,(Griffin, A. M., and Griffin, H. G., eds.), 1994, New Jersey: HumanaPress; von Heinje, G., (1987) Sequence Analysis in Molecular Biology,New York: Academic Press; Sequence Analysis Primer, (Gribskov, M. andDevereux, J., eds.), 1991, New York: M. Stockton Press; and Carillo etal., (1988) SIAM J. Applied Math. 48:1073.

In calculating percent identity, the sequences being compared arealigned in a way that gives the largest match between the sequences. Thecomputer program used to determine percent identity is the GCG programpackage, which includes GAP (Devereux et al., (1984) Nucl. Acid Res.12:387; Genetics Computer Group, University of Wisconsin, Madison,Wis.). The computer algorithm GAP is used to align the two polypeptidesor polynucleotides for which the percent sequence identity is to bedetermined. The sequences are aligned for optimal matching of theirrespective amino acid or nucleotide (the “matched span”, as determinedby the algorithm). A gap opening penalty (which is calculated as 3× theaverage diagonal, wherein the “average diagonal” is the average of thediagonal of the comparison matrix being used; the “diagonal” is thescore or number assigned to each perfect amino acid match by theparticular comparison matrix) and a gap extension penalty (which isusually 1/10 times the gap opening penalty), as well as a comparisonmatrix such as PAM 250 or BLOSUM 62 are used in conjunction with thealgorithm. In certain embodiments, a standard comparison matrix (see,Dayhoff et al., (1978) Atlas of Protein Sequence and Structure 5:345-352for the PAM 250 comparison matrix; Henikoff et al., (1992) Proc. Natl.Acad. Sci. U.S.A. 89:10915-10919 for the BLOSUM 62 comparison matrix) isalso used by the algorithm.

Recommended parameters for determining percent identity for polypeptidesor nucleotide sequences using the GAP program are the following:

Algorithm: Needleman et al., 1970, J. Mol. Biol. 48:443-13;

Comparison matrix: BLOSUM 62 from Henikoff et al., 1992, supra;

Gap Penalty: 12 (but with no penalty for end gaps)

Gap Length Penalty: 4

Threshold of Similarity: 0

Certain alignment schemes for aligning two amino acid sequences canresult in matching of only a short region of the two sequences, and thissmall aligned region can have very high sequence identity even thoughthere is no significant relationship between the two full-lengthsequences. Accordingly, the selected alignment method (e.g., the GAPprogram) can be adjusted if so desired to result in an alignment thatspans at least 50 contiguous amino acids of the target polypeptide.

The term “GDF15 polypeptide” means a naturally-occurring or “wild-type”GDF15 expressed in a mammal, including without limitation, a human,rabbit, monkey (e.g. cynomolgous monkey), dog, rat mouse or pig. In oneaspect, GDF15 polypeptide refers to any full-length GDF15, e.g., SEQ IDNO:4, which consists of 308 amino acid residues and which is encoded bythe nucleotide sequence SEQ ID NO:3; any form comprising the active andprodomains of the polypeptide, e.g., SEQ ID NO:8, which consists of 279amino acid residues and which is encoded by the nucleotide sequence SEQID NO:7, and in which the 29 amino acid residues at the amino-terminalend of the full-length GDF15 (i.e., which constitute the signal peptide)have been removed; and any form of GDF15 comprising the active domainfrom which the prodomain and signal sequence have been removed, e.g.,SEQ ID NO:12, which consists of 112 amino acid residues and which isencoded by the nucleotide sequence SEQ ID NO:11, in which the signalsequence and the prodomain have been removed. GDF15 polypeptides can butneed not comprise an amino-terminal methionine, which may be introducedby engineering or as a result of a bacterial expression process.

The term “GDF15 mutant polypeptide” refers to a GDF15 polypeptide (e.g.,SEQ ID NOs:4, 8 or 12) that has been modified. Such modificationsinclude, but are not limited to, one or more amino acid substitutions,including substitutions with non-naturally-occurring amino acidsnon-naturally-occurring amino acid analogs and amino acid mimetics,additions or deletions. In one aspect, the term “GDF15 mutantpolypeptide” refers to a GDF15 polypeptide in which at least one residuenormally found at a given position of the naturally-occurring GDF15polypeptide is deleted or is replaced by a residue not normally found atthat position in the naturally-occurring GDF15 polypeptide. In somecases it will be desirable to replace a single residue normally found ata given position in the sequence of a naturally-occurring GDF15polypeptide with more than one residue that is not normally found at theposition; in still other cases it may be desirable to maintain thesequence of the naturally-occurring GDF15 polypeptide and insert one ormore residues at a given position in the protein; in still other casesit may be desirable to delete a given residue entirely; all of theseconstructs are encompassed by the term “GDF15 mutant polypeptide.”

In various embodiments, a GDF15 mutant polypeptide comprises an aminoacid sequence that is at least about 85 percent identical to anaturally-occurring GDF15 polypeptide (e.g., SEQ ID NOs:4, 8 or 12). Inother embodiments, a GDF15 mutant polypeptide comprises an amino acidsequence that is at least about 90 percent, or about 95, 96, 97, 98, or99 percent identical to a naturally-occurring GDF15 polypeptide aminoacid sequence (e.g., SEQ ID NOs:4, 8 or 12). Such GDF15 mutantpolypeptides preferably, but need not, possess at least one activity ofa naturally-occurring GDF15 polypeptide, such as the ability to lowerblood glucose, insulin, triglyceride, or cholesterol levels; the abilityto reduce body weight; the ability to improve glucose tolerance, lipidtolerance, or insulin sensitivity; or the ability to lower urine glucoseand protein excretion.

The term “GDF15 region” encompasses GDF15 polypeptides and GDF15 mutantpolypeptides and sequences as defined above.

A GDF15 polypeptide or a GDF15 mutant polypeptide is preferablybiologically active. In some instances, a GDF15 polypeptide or a GDF15mutant polypeptide used to treat or ameliorate a metabolic disorder in asubject from or derived from a different species as the subject. In someinstances, a GDF15 polypeptide or a GDF15 mutant polypeptide used totreat or ameliorate a metabolic disorder in a subject from or derivedfrom the same species as the subject.

The term “native Fc” refers to molecule or sequence comprising thesequence of a non-antigen-binding fragment resulting from digestion ofwhole antibody, whether in monomeric or multimeric form. The originalimmunoglobulin source of the native Fc is preferably of human origin andmay be any of the immunoglobulins, although IgG1 and IgG2 are preferred.Native Fc's are made up of monomeric polypeptides that may be linkedinto dimeric or multimeric forms by covalent (i.e., disulfide bonds) andnon-covalent association. The number of intermolecular disulfide bondsbetween monomeric subunits of native Fc molecules ranges from 1 to 4depending on class (e.g., IgG, IgA, IgE) or subclass (e.g., IgG1, IgG2,IgG3, IgA1, IgGA2). One example of a native Fc is a disulfide-bondeddimer resulting from papain digestion of an IgG (see Ellison et al.(1982), Nucleic Acids Res. 10: 4071-9). The term “native Fc” as usedherein is generic to the monomeric, dimeric, and multimeric forms.

The term “Fc variant” refers to a molecule or sequence that is modifiedfrom a native Fc. Such modifications include, but are not limited to,one or more amino acid substitutions, including substitutions withnon-naturally-occurring amino acids, non-naturally-occurring amino acidanalogs and amino acid mimetics, deletions or additions. Thus, the term“Fc variant” comprises a molecule or sequence that is humanized from anon-human native Fc. Furthermore, a native Fc comprises sites that maybe removed because they provide structural features or biologicalactivity that are not required for the fusion molecules of the presentinvention. Thus, the term “Fc variant” comprises a molecule or sequencethat lacks one or more native Fc sites or residues that affect or areinvolved in (1) disulfide bond formation, (2) incompatibility with aselected host cell (3) N-terminal heterogeneity upon expression in aselected host cell, (4) glycosylation, (5) interaction with complement,(6) binding to an Fc receptor other than a salvage receptor, or (7)antibody-dependent cellular cytotoxicity (ADCC). Fc variants aredescribed in further detail hereinafter. In various embodiments, an Fcvariant comprises an amino acid sequence that is at least about 85percent identical to a native Fc. In other embodiments, an Fc variantcomprises an amino acid sequence that is at least about 90 percent, orabout 95, 96, 97, 98, or 99 percent identical to a native Fc.

The term “Fc domain” encompasses native Fc and Fc variant molecules andsequences as defined above. As with Fc variants and native Fcs, the term“Fc domain” includes molecules in monomeric or multimeric form.

The term “multimer” as applied to Fc domains or molecules comprising Fcdomains refers to molecules having two or more polypeptide chainsassociated covalently, noncovalently, or by both covalent andnon-covalent interactions. IgG molecules typically form dimers; IgM,pentamers; IgD, dimers; and IgA, monomers, dimers, trimers, ortetramers. Multimers may be formed by exploiting the sequence andresulting activity of the native Ig source of the Fc or by derivatizingsuch a native Fc.

The term “dimer” as applied to Fc domains or molecules comprising Fcdomains refers to molecules having two polypeptide chains associatedcovalently, non-covalently or by both covalent and non-covalentinteractions.

The term “hinge” or “hinge region” herein includes the flexiblepolypeptide comprising the amino acids between the first and secondconstant domains of an antibody. The “hinge region” as referred toherein is a sequence region of 6-62 amino acids in length, only presentin IgA, IgD and IgG, which encompasses the cysteine residues that bridgethe two heavy chains.

The “polypeptide linker” refers to a short polypeptide, generally from 1to 30 amino acid residues in length, that covalently links together twopolypeptides, typically via peptide bonds.

As used herein, the term HSA polypeptide encompasses anaturally-occurring “wild type” human serum albumin. The term alsoincludes various bioactive fragments and variants, fusion proteins, andmodified forms of the wild type HSA protein. Such bioactive fragments orvariants, fusion proteins, and modified forms of wild type HSA proteinhave at least a portion of the amino acid sequence of substantialsequence identity to the wild type HAS protein. In certain embodiments,such bioactive fragments or variants, fusion proteins, and modifiedforms of wild type HSA protein have an amino acid sequence that is atleast about 85 percent identical to the wild type HSA. In otherembodiments, such bioactive fragments or variants, fusion proteins, andmodified forms of wild type HSA protein have an amino acid sequence thatis at least about 90 percent, or about 95, 96, 97, 98, or 99 percentidentical to the wild type HAS.

II. Fc Fusions Comprising GDF15 Polypeptides or GDF15 MutantPolypeptides, and Polynucleotides

A range of Fc fusion proteins comprising a GDF15 polypeptide or a GDF15mutant polypeptide are provided herein. In some embodiments, the fusionproteins comprise a native Fc. In some embodiments, the fusion proteinscomprise an Fc domain that has been engineered.

In some embodiments, the GDF15 polypeptide- (or GDF15 mutantpolypeptide-) containing Fc fusion proteins associate with anotherpolypeptide chain consisting of or comprising an Fc domain to form aheterodimer. In some embodiments, two such heterodimers associate toform a heterotetramer. Some of these polypeptide constructs (i.e., GDF15polypeptide- (or GDF15 mutant polypeptide-) containing Fc fusionproteins and multimers comprising one or more such GDF15 polypeptide-(or GDF15 mutant polypeptide-) containing Fc fusion proteins) werestudied empirically, as described in the Examples presented hereinbelow.

Antibodies belong to the immunoglobulin class of proteins which includesIgG, IgA, IgE, IgM, and IgD. The most abundant immunoglobulin class inhuman serum is IgG (Deisenhofer J 1981 Biochem 20:2361-2370; Huber R1984 Behring Inst Mitt 76:1-14; Roux K H 1999 Int Arch Allergy Immunol120:85-99). The IgG structure has four chains, two light and two heavychains; each light chain has two domains and each heavy chain has fourdomains. The antigen binding site is located in the Fab domain (Fragmentantigen binding) which contains a variable light (VL) and a variableheavy (VH) chain domain as well as constant light (LC) and constantheavy (CH1) chain domains. The CH2 and CH3 domain region of the heavychain is called Fc (Fragment crystallizable). The IgG molecule can beconsidered as a heterotetramer having two heavy chains that are heldtogether by disulfide bonds (—S—S—) at the hinge region and two lightchains. The number of hinge disulfide bonds varies among theimmunoglobulin subclasses (Papadea C 1989 Crit Rev Clin Lab Sci27:27-58). The FcRn binding site is located in the Fc domain of theantibody (Martin W L 2001 Mol Cell 7:867-877), and thus the extendedserum half-life property of the antibody is retained in the Fc fragment.The Fc domain alone can be thought of as a homodimer of heavy chainscomprising CH2 and CH3 domains.

In certain preferred embodiments, the fusion proteins described hereincomprise an IgG Fc domain, derived from a wild-type human IgG Fc domain.By “wild-type” human IgG Fc, it is meant a sequence of amino acids thatoccurs naturally within the human population. Of course, just as the Fcsequences may vary slightly between individuals, one or more alterationsmay be made to a wild-type sequence and still remain within the scope ofthe invention. For example, the Fc domain may contain additionalalterations that are not related to the present invention, such as amutation in a glycosylation site or the inclusion of an unnatural aminoacid. In certain embodiments, the polypeptide containing the CH3 regionis an IgG molecule and further contains a CH1 and CH2 domain. Exemplaryhuman IgG sequences comprise the constant regions of IgG1, IgG2, IgG3and IgG4. The Fc domain also may be comprised within the constant regionof an IgA, IgD, IgE, and IgM heavy chain.

Some Fc fusion proteins containing GDF15 polypeptide or GDF15 mutantpolypeptide, and multimers comprising such Fc fusion proteins includethose described below.

II.A. DhMonoFc Constructs

The designations “Mono-” or “MonoFc domain” in the instant disclosurerefer to an Fc domain that has been engineered to reduce or prevent theformation of homodimers. In one embodiment, a MonoFc domain is providedby introducing a tyrosine to threonine mutation (Y349T) and two lysineto aspartic acid mutations (K392D and K409D) in a native Fc or Fcvariant.

The C-terminal lysine (K447) optionally may be deleted in the MonoFc.This may be advantageous, for example, when a peptide is fused at theC-terminus to reduce proteolysis of the fusion protein.

A fusion protein is provided comprising a MonoFc domain and a GDF15region. Typically, the N-terminus of the GDF15 region is linked,directly or via a polypeptide linker, to the C-terminus of the MonoFcdomain. However, in some embodiments, the N-terminus of the MonoFcdomain is linked, directly or via a polypeptide linker, to theC-terminus of the GDF15 region.

In certain embodiments, a dimer is provided comprising two such fusionproteins linked via an interchain disulfide bond between theirrespective GDF15 regions. In some embodiments the dimer is a homodimer.In other embodiments, the dimer is a heterodimer.

The designations “DhMono-” or “DhMonoFc domain” in the instantdisclosure refer to an Fc domain from which all or part of the hingeregion has been removed that has been engineered to reduce or preventthe formation of homodimers. In one embodiment, a DhMonoFc domain isprovided introducing a tyrosine to threonine mutation (Y349T) and twolysine to aspartic acid mutations (K392D and K409D) in a native Fc or Fcvariant from which all or part of the hinge region has been removed.

The C-terminal lysine (K447) optionally may be deleted in the DhMonoFc.This may be advantageous, for example, when a peptide is fused at theC-terminus to reduce proteolysis of the fusion protein.

A fusion protein is provided comprising a DhMonoFc domain and a GDF15region. Typically, the N-terminus of the GDF15 region is linked,directly or via a polypeptide linker, to the C-terminus of the DhMonoFcdomain. However, in some embodiments, the N-terminus of the DhMonoFcdomain is linked, directly or via a polypeptide linker, to theC-terminus of the GDF15 region.

In certain embodiments, a dimer is provided comprising two such fusionproteins linked via an interchain disulfide bond between theirrespective GDF15 regions. In some embodiments the dimer is a homodimer.In other embodiments, the dimer is a heterodimer.

II.A.1 DhMonoFc-GDF15

The designation “MonoFc-GDF15” in the instant disclosure refers to afusion protein comprising a GDF15 polypeptide, the N-terminus of whichis linked directly to the C-terminus of a MonoFc domain.

In certain embodiments, a homodimer is provided comprising two suchfusion proteins linked via an interchain disulfide bond between theirrespective GDF15 regions.

More particularly, in a specific embodiment, the homodimer comprises:

(a) two MonoFc domains (one each monomer) comprising the sequence:

(SEQ ID NO: 22) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVTTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPG,and

(b) two GDF15 polypeptides (one each monomer) comprising the sequence ofSEQ ID NO:12.

In a preferred embodiment, the fusion protein comprises the amino acidsequence:

(SEQ ID NO: 46) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVTTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 45) gcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgaccaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtgcgcgcaacggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcata.

As discussed above, in a specific embodiment, a homodimer is providedcomprising two monomers having the sequence of SEQ ID NO:46.

II.A.2 DhMonoFc-(G₄S)₄-GDF15

The designation “DhMonoFc-(G₄S)₄-GDF15” in the instant disclosure refersto a fusion protein comprising a GDF15 polypeptide linked to a DhMonoFcdomain via a polypeptide linker comprising the sequence of SEQ ID NO:18that connects the N-terminus of the GDF15 polypeptide to the C-terminusof the DhMonoFc domain.

In certain embodiments, a homodimer is provided comprising two suchfusion proteins linked via an interchain disulfide bond between theirrespective GDF15 regions.

More particularly, in a specific embodiment, the homodimer comprises:

(a) two DhMonoFc domains (one each monomer) comprising the sequence ofSEQ ID NO:22,

(b) two GDF15 polypeptides (one each monomer) comprising the sequence ofSEQ ID NO:12, and

(c) two polypeptide linkers (one each monomer) comprising the sequence:

GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:18)

each linking the N-terminus of a GDF15 polypeptide to the C-terminus ofa DhMonoFc domain.

In a preferred embodiment, the fusion protein comprises the amino acidsequence (linker sequence double underlined):

(SEQ ID NO: 24) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVTTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 23) cacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgaccaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtggaggtggtggatccggaggcggtggaagcggaggtggtggatctggaggcggtggaagcgcgcgcaacggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcatatga.

As discussed above, in a specific embodiment, a homodimer is providedcomprising two monomers having the sequence of SEQ ID NO:24.

II.A.3 DhMonoFc-(G₄S)₄-GDF15(H6D)

The designation “DhMonoFc-(G₄S)₄-GDF15(H6D)” in the instant disclosurerefers to fusion protein comprising a GDF15 polypeptide linked to aDhMonoFc domain via a polypeptide linker comprising the sequence of SEQID NO:18 that connects the N-terminus of the GDF15 polypeptide to theC-terminus of the DhMonoFc domain. The GDF15(H6D) variant is anaturally-occurring human GDF15 variant.

In certain embodiments, a homodimer is provided comprising two suchfusion proteins linked via an interchain disulfide bond between theirrespective GDF15 regions.

More particularly, in a specific embodiment, the homodimer comprises:

(a) two DhMonoFc domains (one each monomer) comprising the sequence ofSEQ ID NO:22,

(b) two GDF15(H6D) polypeptides (one each monomer) comprising thesequence:

(SEQ ID NO: 25) ARNGDDCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQT YDDLLAKDCHCI,and

(c) two polypeptide linkers (one each monomer) comprising the sequenceof SEQ ID NO:18 each linking the N-terminus of the GDF15 polypeptide tothe C-terminus of the DhMonoFc domain.

In a preferred embodiment, the fusion protein comprises the amino acidsequence (linker sequence double underlined):

(SEQ ID NO: 27) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVTTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSARNGDDCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 26) gcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgaccaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtggaggtggtggatccggaggcggtggaagcggaggtggtggatctggaggcggtggaagcgcgcgcaacggagacgactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcatatga.

As discussed above, in a specific embodiment, a homodimer is providedcomprising two monomers having the sequence of SEQ ID NO:27.

II.B. HemiFc

The designations “Hemi-” or “HemiFc domain” in the instant disclosurerefer to a polypeptide chain comprising a first Fc domain linked,directly or via a polypeptide linker, to a second Fc domain. In oneembodiment, the first Fc domain and the second Fc domain have the samesequence. In another embodiment, the first Fc domain and second Fcdomain have different sequences. Typically, the first and second Fcdomains are covalently associated via disulfide bonds between theirrespective hinge regions.

The C-terminal lysine (K447) optionally may be deleted in the first Fcdomain, the second Fc domain, or both. This may be advantageous, forexample, when a peptide is fused at the C-terminus to reduce proteolysisof the fusion protein.

A fusion protein is provided comprising a HemiFc domain and a GDF15region. Typically, the N-terminus of the GDF15 region is linked,directly or via a polypeptide linker, to the C-terminus of the HemiFcdomain. However, in some embodiments, the N-terminus of the HemiFcdomain is linked, directly or via a polypeptide linker, to theC-terminus of the GDF15 region.

In certain embodiments, a dimer is provided comprising two such fusionproteins linked via an interchain disulfide bond between theirrespective GDF15 regions. In some embodiments the dimer is a homodimer.In other embodiments, the dimer is a heterodimer. See FIG. 3 for agraphic depiction of an embodiment of a homodimer of twoGGG-Fc-(G₄S)₄-Fc-S(G₄S)₄-GDF15 fusion proteins.

GGGFc-(G₄S)₄-Fc-S(G₄S)₄-GDF15

The designation “GGGFc-(G₄S)₄-Fc-S(G₄S)₄-GDF15” in the instantdisclosure refers to a fusion protein comprising a GDF15 polypeptidelinked to HemiFc domain via a first polypeptide linker comprising thesequence of SEQ ID NO:30, that connects the N-terminus of the GDF15polypeptide to the C-terminus of the HemiFc domain. The HemiFc domaincomprises a first Fc domain linked to a second Fc domain via apolypeptide linker comprising the sequence of SEQ ID NO:18, thatconnects the N-terminus of the first Fc domain to the C-terminus of thesecond Fc domain (which has three glycine residues added to itsN-terminus).

In certain embodiments, a homodimer is provided comprising two suchfusion proteins linked via an interchain disulfide bond between theirrespective GDF15 regions.

More particularly, in a specific embodiment, the homodimer comprises:

(a) two second Fc domains (one each monomer) comprising the sequence(part of the hinge region in parentheses):

(SEQ ID NO: 28) GGG(ERKSSVECPPCP)APPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG,

(b) two first Fc domains (one each monomer) comprising the sequence(part of the hinge region in parentheses):

(SEQ ID NO: 29) (ERKSSVECPPCP)APPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG.

(c) two second polypeptide linkers (one each monomer) comprising thesequence of SEQ ID NO:18 linking the N-terminus of the first Fc domainto the C-terminus of the second Fc domain,

(d) two GDF15 polypeptides (one each monomer) comprising the sequence ofSEQ ID NO:12, and

(e) two first polypeptide linkers (one each monomer) comprisingsequence:

SGGGGSGGGGSGGGGSGGGGS (SEQ ID NO:30)

linking the N-terminus of a GDF15 polypeptide to the C-terminus of afirst Fc domain.

In a preferred embodiment, the fusion protein comprises the amino acidsequence (part of the hinge region in parentheses; linker sequencesdouble underlined):

(SEQ ID NO: 32) GGG(ERKSSVECPPCP)APPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGS(ERRSSVECPPCP)APPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSGGGGSGGGGSARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSL QTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 31) ggaggtggagagcgcaaatcttctgtcgagtgcccaccgtgcccagcaccacctgtggcaggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccacgaagaccccgaggtccagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaaaccacgggaggagcagttcaacagcacgttccgtgtggtcagcgtcctcaccgttgtgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccagcccccatcgagaaaaccatctccaaaaccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacacctcccatgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtggaggtggcggtagcggtggcggaggttcaggtggcggcggaagcggtggaggaggttcagagcggaaatccagcgttgaatgtcctccgtgccctgctccacccgtcgcggggcctagtgtcttccttttccctccaaaaccaaaggatacactgatgatcagccggacccccgaggttacgtgcgtcgtcgtcgatgtctcccacgaggatccagaggtccaattcaactggtacgtggacggggtcgaggtgcataatgcaaagacaaagccacgggaagagcagtttaactctactttccgcgtggtttctgtgctgaccgtggtgcaccaagattggctcaacggcaaggagtacaagtgcaaggtaagcaataaggggctccctgcccccattgagaagactatctccaagacaaagggacagccacgcgagccacaagtctatacactccccccttcccgcgaagaaatgaccaagaatcaggttagcctgacatgcttggttaagggtttctacccctctgacatagccgtggagtgggagagcaatggacaaccagagaacaactacaagaccaccccacccatgctggatagcgacggttcattctttctgtatagtaagcttaccgtggacaagtcccggtggcaacaaggaaatgtcttttcatgctctgtgatgcacgaggccttgcataatcactatactcagaagagcttgagcctcagccccggatctggaggtggcggatccgggggcggtggaagcggaggtggtggatcgggaggcggtggaagcgcgcgcaacggcgaccactgtccgctcgggcccggacgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcatatga.

As discussed above, in a specific embodiment, a homodimer is providedcomprising two monomers having the sequence of SEQ ID NO:32.

II.C. DhHemiFc

The designations “DhHemi-” or “DhHemiFc domain” in the instantdisclosure refer to a polypeptide chain comprising a first Fc domainfrom which all or part of the hinge region has been removed (“DhFc”domain) linked, directly or via a polypeptide linker, to a second DhFcdomain. In particular embodiments, the N-terminal 12 amino acids in thehinge region are removed, e.g., ERKSSVECPPCP (SEQ ID NO:15). In oneembodiment, the first DhFc domain and the second DhFc domain have thesame sequence. In another embodiment, the first DhFc domain and thesecond DhFc domain have different sequences.

A fusion protein is provided comprising a DhHemiFc domain and a GDF15region. Typically, the N-terminus of the GDF15 region is linked,directly or via a polypeptide linker, to the C-terminus of the DhHemiFcdomain. However, in some embodiments, the N-terminus of the DhHemiFcdomain is linked, directly or via a polypeptide linker, to theC-terminus of the GDF15 region.

In certain embodiments, a dimer is provided comprising two such fusionproteins linked via an interchain disulfide bond between theirrespective GDF15 regions. In some embodiments the dimer is a homodimer.In other embodiments, the dimer is a heterodimer.

GGGDhFc-(G₄S)₅-DhFc-S(G₄S)₄-GDF15

The designation “GGGDhFc-(G₄S)₅-DhFc-S(G₄S)₄-GDF15” in the instantdisclosure refers to a fusion protein comprising a GDF15 polypeptidelinked to a DhHemiFc domain via a first polypeptide linker comprisingthe sequence of SEQ ID NO:30, that connects the N-terminus of the GDF15polypeptide to the C-terminus of the HemiFc domain. The HemiFc domaincomprises a first DhFc domain linked to a second DhFc domain via apolypeptide linker comprising the sequence of SEQ ID NO:34, thatconnects the N-terminus of the first DhFc domain to the C-terminus ofthe second DhFc domain (which has three glycine residues added to itsN-terminus).

In certain embodiments, a homodimer is provided comprising two suchfusion proteins linked via an interchain disulfide bond between theirrespective GDF15 regions.

More particularly, in a specific embodiment, the homodimer comprises:

(a) two second DhFc domains (one each monomer) comprising the sequence:

(SEQ ID NO: 33) GGGAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPG,

(b) two first DhFc domains (one each monomer) comprising the sequence:

(SEQ ID NO: 35) APPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPG,

(c) two second polypeptide linkers (one each monomer) comprising thesequence:

GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO:34)

linking the N-terminus of the first DhFc domain to the C-terminus of thesecond DhFc domain,

(d) two GDF15 polypeptides (one each monomer) comprising the sequence ofSEQ ID NO:12, and

(e) two first polypeptide linkers (one each monomer) comprising thesequence of SEQ ID NO:30 linking the N-terminus of a GDF15 polypeptideto the C-terminus of a first DhFc domain.

In a preferred embodiment, the fusion protein comprises the amino acidsequence (linker sequences double underlined):

(SEQ ID NO: 37) GGGAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSGGGGSAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSGGGGSGGGGSARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 36) ggcggtggagctccgccggtggctggaccctcagtgttcctctttccaccgaagccgaaggacacccttatgattagccggaccccagaggtcacttgcgtcgtcgtggacgtgtcccatgaggatcccgaagtgcagtttaactggtatgtggacggagtggaggtccataacgccaagaccaagccaagggaagaacagttcaatagcaccttccgggtggtgtccgtgctcaccgtggtgcatcaagactggctgaatggcaaagagtacaaatgtaaggtgtcaaacaaggggctcccagcccctattgaaaagaccatctcaaagactaagggacagccacgcgaacctcaagtgtataccctcccgccttcacgcgaagaaatgactaagaatcaggtcagccttacttgtctggtcaagggcttctacccgagcgacattgcagtcgaatgggagagcaatggtcagccagagaataactacaagaccactcctcccatgcttgatagcgatggaagctttttcctttacagcaagcttactgtggataagtctcgctggcaacagggaaatgtgttcagctgttcagtgatgcatgaagcactccacaatcattacacccagaagtcactcagcctctcacccggaggaggaggcggttctggtggaggagggtctggaggtggagggagcggcggaggcgggtctggcggtggtgggtctgagaggaagtcatcagtggaatgcccaccatgccctgctcctcccgtggccggtccgagcgtgtttctcttcccacctaagcccaaggacactctgatgatctcacggactccggaagtgacttgtgtggtggtggacgtgtctcatgaggaccctgaagtgcagttcaactggtacgtggacggcgtggaggtgcacaatgctaagaccaagcctagagaggaacagttcaattccacctttcgcgtggtgagcgtcctgaccgtcgtgcaccaggactggcttaacggaaaggaatacaagtgcaaggtgtccaacaaaggccttccagctcccattgagaaaaccatctctaaaactaagggtcaaccaagggaaccccaagtctacaccctccctccgtctagagaagagatgaccaaaaaccaggtgtccctgacctgtctggtgaagggattttacccctcagacatcgccgtggagtgggaaagcaacggacagcccgaaaacaactataagactacccctcctatgctggactcagacggatctttcttcctctatagcaagctcactgtggacaaatccagatggcaacaagggaatgtgttctcatgcagcgtgatgcacgaggctcttcacaaccactatacccagaagagcctgtctctttcacctggttccggaggtggtgggagcggagggggtggatcaggtggtggagggtccggaggcggaggatccgcacggaatggcgaccactgtccactgggacccggaagatgttgtcgcctccacaccgtgagggcctctctggaggaccttggctgggccgactgggtcctgtcacctcgggaggtccaagtcaccatgtgtatcggagcctgccccagccaattcagagcagcaaatatgcacgcacagattaagaccagcctgcatcggcttaaacctgatactgtgccggctccttgttgcgtgccagcatcttacaacccgatggtgctgatccagaaaaccgataccggtgtctccctccagacttacgacgacctccttgcaaaggactgc cattgcatc.

As discussed above, in a specific embodiment, a homodimer is providedcomprising two monomers comprising the sequence of SEQ ID NO:37.

II.D. Knob/Hole

The designations “knob-” or “knobFc domain” in the instant disclosurerefer to an Fc domain comprising a “knob” mutation. The designations“hole-” or “holeFc domain” in the instant disclosure refers to a nativeFc or Fc variant comprising a “hole” mutation.

“Knobs” may be created by replacing small amino acid side chains withlarger ones and “holes” may be created by replacing large amino acidside chains with smaller ones. See, e.g., Ridgway J B B 1996, ProteinEng. 9:617-621; Merchant A M 1998, Nature Biotech. 16:677-681.

In one embodiment, a “knobFc” domain is provided by introducing athreonine to tryptophan mutation (T366W) in the sequence of an Fcdomain. In one embodiment, a “holeFc” domain is provided by introducinga threonine to serine mutation (T366S), a leucine to alanine mutation(L368A) and a tyrosine to valine mutation (Y407V) in the sequence of anFc domain.

The C-terminal lysine (K447) optionally may be deleted in the knobFcdomain, the holeFc domain, or both. This may be advantageous, forexample, when a peptide is fused at the C-terminus to reduce proteolysisof the fusion protein.

In one embodiment, a “DhknobFc” domain is provided by introducing athreonine to tryptophan mutation (T366W) in the sequence of an Fc domainfrom which all or part of the hinge region has been removed. In oneembodiment, a “DhholeFc” domain is provided by introducing a threonineto serine mutation (T366S), a leucine to alanine mutation (L368A) and atyrosine to valine mutation (Y407V) in the sequence of an Fc domain fromwhich all or part of the hinge region has been removed.

The C-terminal lysine (K447) optionally may be deleted in the DhknobFc,the DhholeFc, or both. This may be advantageous, for example, when apeptide is fused at the C-terminus to reduce proteolysis of the fusionprotein.

In some embodiments, a heterodimer is providing comprising (i) a firstpolypeptide chain comprising a GDF15 region linked to a holeFc domaindirectly or via a polypeptide linker and (ii) a second polypeptide chaincomprising a knobFc domain. In other embodiments, a heterodimer isprovided comprising (i) a first polypeptide chain comprising a GDF15region linked to a knobFc domain, directly or via a polypeptide linkerand (ii) a second polypeptide chain comprising a holeFc domain.

In some embodiments, a heterodimer is providing comprising (i) a firstpolypeptide chain comprising a GDF15 region linked to a DhholeFc domaindirectly or via a polypeptide linker and (ii) a second polypeptide chaincomprising a DhknobFc domain. In other embodiments, a heterodimer isprovided comprising (i) a first polypeptide chain comprising a GDF15region linked to a DhknobFc domain, directly or via a polypeptide linkerand (ii) a second polypeptide chain comprising a DhholeFc domain.

In some embodiments, a tetramer is provided comprising two suchheterodimers in which the heterodimers are linked via an interchaindisulfide bond between the GDF15 regions of their respective firstpolypeptide chains. See FIG. 1 for a graphic depiction of an embodimentof a heterotetramer comprising two heterodimers, where each heterodimercomprises (i) a first polypeptide chain comprising a GDF15 polypeptidelinked to a DhknobFc domain via a polypeptide linker and (ii) a secondpolypeptide chain comprising a DhholeFc domain.

DhknobFc-(G₄S)₄-GDF15:DhholeFc

The designation “DhknobFc-(G₄S)₄-GDF15:DhholeFc” in the instantdisclosure refers to a heterodimer comprising (i) a first polypeptidechain comprising a GDF15 polypeptide linked to a DhknobFc domain via alinker comprising the sequence of SEQ ID NO:18 that connects theN-terminus of the GDF15 polypeptide to the C-terminus of the DhknobFcdomain and (ii) a polypeptide chain comprising a DhholeFc domain.

In certain embodiments, a tetramer is provided comprising a dimer of twoDhknobFc-(G₄S)₄-GDF15:DhholeFc heterodimers in which the firstpolypeptide chains of each heterodimer are linked via an interchaindisulfide bond between their respective GDF15 regions.

More particularly, in a specific embodiment, the tetramer comprises:

(a) two DhknobFc domains (one each heterodimer) comprising the sequence:

(SEQ ID NO: 16) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPG,

(b) two DhholeFc domains (one each heterodimer) comprising the sequence:

(SEQ ID NO: 281) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPG,

(c) two GDF15 polypeptides (one each heterodimer) comprising thesequence of SEQ ID NO:12, and

(d) two polypeptide linkers (one each heterodimer) comprising thesequence of SEQ ID NO:18 each linking the N-terminus of a GDF15polypeptide to the C-terminus of a DhknobFc domain via peptide bonds.

In a preferred embodiment, the first polypeptide chain comprises theamino acid sequence (linker sequence double underlined):

(SEQ ID NO: 20) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 19) gcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgtggtgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtggaggtggtggatccggaggcggtggaagcggaggtggtggatctggaggcggtggaagcgcgcgcaacggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcatatga.In a preferred embodiment, the second polypeptide chain comprises theamino acid sequence:

(SEQ ID NO: 17) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 21) gcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgagctgcgcggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctcgtcagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaa atga

As discussed above, in a specific embodiment, a tetramer is providedcomprising two monomers having the sequence of SEQ ID NO:20 and twomonomers having the sequence of SEQ ID NO:17.

II.E. Charged Pair (delHinge)

The designation “CpmFc(+) domain” in the instant disclosure refers to anFc domain comprising a “positive” charged pair mutation. The designation“CpmFc(−) domain” in the instant disclosure refers to an Fc domaincomprising a “negative” charged pair mutation. Note that use of theterms “positive” and “negative” is for ease of reference (i.e., todescribe the nature of the charged pair mutations in the Fc domains) andnot to indicate that the overall sequence or construct necessarily has apositive or negative charge.

In one embodiment, a “positive” charged pair mutation is provided byintroducing a glutamic acid to lysine mutation (E356K) and an asparticacid to lysine mutation (D399K) in the sequence of an Fc domain. In oneembodiment, a “negative” charged pair mutation is provided byintroducing two lysine to aspartic acid mutations (K392D, K409D) in thesequence of an Fc domain.

When incubated together, the aspartate residues associate with thelysine residues through electrostatic force, facilitating formation ofheterodimers between the CpmFc(+) domains and CpmFc(−) domains, andreducing or preventing formation of homodimers between the CpmFc(+)sequences or between CpmFc(−) sequences.

The C-terminal lysine (K447) optionally may be deleted in the CpmFc(+)domain, the CpmFc(−) domain, or both. This may be advantageous, forexample, when a peptide is fused at the C-terminus to reduce proteolysisof the fusion protein.

In one embodiment, a “DhCpmFc(+)” domain is provided by introducing aglutamic acid to lysine mutation (E356K) and an aspartic acid to lysinemutation (D399K) in the sequence of an Fc domain from which all or partof the hinge region has been removed. In one embodiment, a “DhCpmFc(−)”domain is provided by introducing two lysine to aspartic acid mutations(K392D, K409D) in the sequence of an Fc domain from which all or part ofthe hinge region has been removed.

When incubated together, the aspartate residues associate with thelysine residues through electrostatic force, facilitating formation ofheterodimers between the DhCpmFc(+) domains and DhCpmFc(−) domains, andreducing or preventing formation of homodimers between the DhCpmFc(+)sequences or between DhCpmFc(−) sequences.

The C-terminal lysine (K447) optionally may be deleted in theDhCpmFc(+), the DhCpmFc(−), or both. This may be advantageous, forexample, when a peptide is fused at the C-terminus to reduce proteolysisof the fusion protein.

In some embodiments, a heterodimer is provided comprising (i) a firstpolypeptide chain comprising a GDF15 region linked to a CpmFc(+) domain,and (ii) a second polypeptide chain comprising a CpmFc(−) domain. Insome embodiments, the N-terminus of the GDF15 region is linked to theC-terminus of the CpmFc(+) domain, directly or via a polypeptide linker.In other embodiments, the N-terminus of the CpmFc(+) domain is linked tothe C-terminus of the GDF15 region, directly or via a polypeptidelinker.

In other embodiments, a heterodimer is provided comprising (i) a firstpolypeptide chain comprising a GDF15 region linked to a CpmFc(−) domain,and (ii) a second polypeptide chain comprising a CpmFc(+) domain. Insome embodiments, the N-terminus of the GDF15 region is linked to theC-terminus of the CpmFc(−) domain, directly or via a polypeptide linker.In other embodiments, the N-terminus of the CpmFc(−) domain is linked tothe C-terminus of the GDF15 region, directly or via a polypeptidelinker.

In some embodiments, a heterodimer is provided comprising (i) a firstpolypeptide chain comprising a GDF15 region linked to a DhCpmFc(+)domain, and (ii) a second polypeptide chain comprising a DhCpmFc(−)domain. In some embodiments, the N-terminus of the GDF15 region islinked to the C-terminus of the DhCpmFc(+) domain, directly or via apolypeptide linker. In other embodiments, the N-terminus of theDhCpmFc(+) domain is linked to the C-terminus of the GDF15 region,directly or via a polypeptide linker.

In other embodiments, a heterodimer is provided comprising (i) a firstpolypeptide chain comprising a GDF15 region linked to a DhCpmFc(−)domain, and (ii) a second polypeptide chain comprising a DhCpmFc(+)domain. In some embodiments, the N-terminus of the GDF15 region islinked to the C-terminus of the DhCpmFc(−) domain, directly or via apolypeptide linker. In other embodiments, the N-terminus of theDhCpmFc(−) domain is linked to the C-terminus of the GDF15 region,directly or via a polypeptide linker.

In some embodiments, a tetramer is provided comprising a dimer of twosuch heterodimers in which the two first polypeptide chains of theheterodimers are linked via an interchain disulfide bond between theirrespective GDF15 regions. See FIG. 4 for a graphic depiction of anembodiment of a tetramer comprising two heterodimers, where eachheterodimer comprises (i) a first polypeptide chain comprising a GDF15polypeptide linked via a polypeptide linker to a DhCpmFc(−) domain and(ii) a second polypeptide chain comprising a DhCpmFc(+) domain.

II.E.1 DhCpmFc(+)-(1K)-GDF15:DhCpmFc(−)

The designation “DhCpmFc(+)-(1K)-GDF15:DhCpmFc(−)” in the instantdisclosure refers to a heterodimer comprising (i) a first polypeptidechain comprising a GDF15 polypeptide linked to a DhCpmFc(+) domain via alinker comprising the sequence of SEQ ID NO:40 that connects theN-terminus of the GDF15 polypeptide to the C-terminus of the DhCpmFc(+)domain and (ii) a second polypeptide chain comprising a DhCpmFc(−)domain.

In certain embodiments, a tetramer is provided comprising a dimer of twoDhCpmFc(+)-(1K)-GDF15:DhCpmFc(−) heterodimers in which the two firstpolypeptide chains of each heterodimer are linked via an interchaindisulfide bond between their respective GDF15 regions.

More particularly, in a specific embodiment, the tetramer comprises:

(a) two DhCpmFc(+) domains (one each heterodimer) comprising thesequence:

(SEQ ID NO: 38) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPG,

(b) two DhCpmFc(−) domains (one each heterodimer) comprising thesequence:

(SEQ ID NO: 282) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPG,

(c) two GDF15 polypeptides (one each heterodimer) comprising thesequence of SEQ ID NO:12, and

(d) two polypeptide linkers (one each heterodimer) comprising thesequence:

GSGSATGGSGSVASSGSGSATHL (SEQ ID NO:40)

each linking the N-terminus of a GDF15 polypeptide to the C-terminus ofa DhCpmFc(+) domain via a peptide bond.

In a preferred embodiment, the first polypeptide chain comprises theamino acid sequence (linker sequence double underlined):

(SEQ ID NO: 42) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGSGSATGGSGSVASSGSGSATHLARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHC I,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 41) gccccagagctgcttggtggaccatccgtgttcctgtttcctccaaagccgaaggacaccctgatgatctcaagaactccggaagtgacttgcgtcgtcgtggacgtgtcacatgaggatccagaggtcaagttcaattggtatgtggacggagtggaagtgcataacgccaagaccaaaccccgcgaagaacagtacaatagcacctaccgcgtggtgagcgtccttactgtgctccaccaggactggcttaatgggaaggaatacaagtgtaaggtgtccaacaaggccctccccgctcccatcgaaaagaccatctcaaaggcaaaggggcaaccaagggaacctcaagtgtacaccctgcctccgagcaggaaggagatgaccaagaaccaggtcagcctgacttgtctcgtgaagggcttctatcccagcgatattgctgtggaatgggagtcaaatggccagcccgagaataactacaaaactaccccacccgtgctgaaatctgatgggtccttcttcctttactccaagctgaccgtggacaagagccgctggcaacaaggcaatgtctttagctgctcagtgatgcatgaggctctccataatcactacactcagaagtcactgtccctgtcacctggcggatccggttctgctactggtggttccggctccgtcgcaagctctggttcaggcagtgcgactcatctggcacggaacggggaccattgtcccctgggacctggtcggtgctgccggcttcacaccgtcagagcctctctggaggaccttggatgggctgattgggtgctgagccctcgggaggtgcaagtcaccatgtgcatcggggcctgccctagccagttccgcgcagccaacatgcacgctcagatcaaaacctctcttcacagactgaagcccgacaccgtgccagcaccttgctgtgtgccggcctcttataaccccatggtcctcattcagaaaaccgacaccggagtgtcacttcagacttacgatgacctcctggccaaggactgccactgc ata.

In a preferred embodiment, the second polypeptide chain and comprisesthe amino acid sequence

(SEQ ID NO: 39) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 43) gcgccggaactgctgggcggcccgagcgtgtttctgtttccgccgaaaccgaaagataccctgatgattagccgcaccccggaagtgacctgcgtggtggtggatgtgagccatgaagatccggaagtgaaatttaactggtatgtggatggcgtggaagtgcataacgcgaaaaccaaaccgcgcgaagaacagtataacagcacctatcgcgtggtgagcgtgctgaccgtgctgcatcaggattggctgaacggcaaagaatataaatgcaaagtgagcaacaaagcgctgccggcgccgattgaaaaaaccattagcaaagcgaaaggccagccgcgcgaaccgcaggtgtataccctgccgccgagccgcgaagaaatgaccaaaaaccaggtgagcctgacctgcctggtgaaaggcttttatccgagcgatattgcggtggaatgggaaagcaacggccagccggaaaacaactatgataccaccccgccggtgctggatagcgatggcagcttttttctgtatagcgatctgaccgtggataaaagccgctggcagcagggcaacgtgtttagctgcagcgtgatgcatgaagcgctgcataaccattatacccagaaaagcctgagcctgagcccgggcaa a.

As discussed above, a tetramer is provided comprising two polypeptidechains comprising the sequence of SEQ ID NO:42 and two polypeptidechains comprising the sequence of SEQ ID NO:39.

II.E.2 DhCpmFc(−)-GDF15:DhCpmFc(+)

The designation “DhCpmFc(−)-GDF15:DhCpmFc(+)” in the instant disclosurerefers to a heterodimer comprising (i) a first polypeptide chaincomprising a GDF15 polypeptide, the N-terminus of which is linkeddirectly to the C-terminus of a DhCpmFc(−) domain, and (ii) a secondpolypeptide chain comprising a DhCpmFc(+) domain.

In certain embodiments, a tetramer is provided comprising a dimer of twoDhCpmFc(−)-GDF15:DhCpmFc(+) heterodimers in which the two firstpolypeptide chains of each heterodimer are linked via an interchaindisulfide bond between their respective GDF15 regions.

More particularly, in a specific embodiment, the tetramer comprises:

(a) two DhCpmFc(+) domains (one each heterodimer) comprising the aminoacid sequence:

(SEQ ID NO: 283) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPG,

(b) two DhCpmFc(−) domains (one each heterodimer) comprising the aminoacid sequence:

(SEQ ID NO: 48) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPG,and,

(c) two GDF15 polypeptides (one each heterodimer) comprising thesequence of SEQ ID NO:12.

In a preferred embodiment, the first polypeptide chain comprises theamino acid sequence:

(SEQ ID NO: 50) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 49) gcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtgcgcgcaacggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcata.

In a preferred embodiment, the second polypeptide chain comprises theamino acid sequence:

(SEQ ID NO: 47) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 51) gcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccggaaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctgaagtccgacggctccttcttcctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaa atga.

As discussed above, a tetramer is provided comprising two polypeptidechains having the sequence of SEQ ID NO:50 and two polypeptide chainshaving the sequence of SEQ ID NO:47.

II.E.3 DhCpmFc(−)-GDF15(N3D):DhCpmFc(+)

The designation “DhCpmFc(−)-GDF15(N3D):DhCpmFc(+)” in the instantdisclosure refers to a heterodimer comprising (i) a first polypeptidechain comprising a naturally-occurring variant of a GDF15 having anasparagine to aspartic acid mutation (N3D) (“GDF15(N3D)”), theN-terminus of which is linked directly to the C-terminus of a DhCpmFc(−)domain, and (ii) a second polypeptide chain comprising a DhCpmFc(+)domain. The N3D mutation may reduce deamidation induced heterogeneity.

In certain embodiments, a tetramer is provided comprising a dimer of twoDhCpmFc(−)-GDF15(N3D):DhCpmFc(+) heterodimers in which the two firstpolypeptide chains of each heterodimer are linked via an interchaindisulfide bond between their respective GDF15 regions.

More particularly, in a specific embodiment, the tetramer comprises:

(a) two DhCpmFc(+) domains (one each heterodimer) having the sequence ofSEQ ID NO:283,

(b) two DhCpmFc(−) domains (one each heterodimer) having the sequence ofSEQ ID NO:48, and

(c) two GDF15(N3D) polypeptides (one each heterodimer) comprising thesequence:

(SEQ ID NO: 52) ARDGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQT YDDLLAKDCHCI.

In a preferred embodiment, the first polypeptide chain comprises theamino acid sequence:

(SEQ ID NO: 54) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGARDGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 53) gcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtgcgcgcgacggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcata.

In a preferred embodiment, the second polypeptide chain comprises theamino acid sequence of SEQ ID NO:47, which is encoded by the nucleicacid sequence of SEQ ID NO:51.

As discussed above, a tetramer is provided comprising two polypeptidechains having the sequence of SEQ ID NO:54 and two polypeptide chainshaving the sequence of SEQ ID NO:47.

II.E.4 DhCpmFc(−)-GDF15(Ndel3):DhCpmFc(+)

The designation “DhCpmFc(−)-GDF15(Ndel3):DhCpmFc(+)” in the instantdisclosure refers to a heterodimer comprising (i) a first polypeptidechain comprising a variant of GDF15 in which the first three amino acidsare deleted (“GDF15(Ndel3”), the N-terminus of which is linked directlyto the C-terminus of a DhCpmFc(−) domain, and (ii) a second polypeptidechain comprising a DhCpmFc(+) domain. The Ndel3 variant may reduce N3deamidation and subsequent D3 isomerization induced heterogeneity.

In certain embodiments, a tetramer is provided, comprising a dimer oftwo DhCpmFc(−)-GDF15(Ndel3):DhCpmFc(+) heterodimers in which the twofirst polypeptide chains of each heterodimer are linked via aninterchain disulfide bond between their respective GDF15 regions.

More particularly, in a specific embodiment, the tetramer comprises:

(a) two DhCpmFc(+) domains (one each heterodimer) having the sequence ofSEQ ID NO:283,

(b) two DhCpmFc(−) domains (one each heterodimer) having the sequence ofSEQ ID NO:48, and

(c) two GDF15(Ndel3) polypeptides (one each heterodimer) having thesequence:

(SEQ ID NO: 55) GDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDD LLAKDCHCI.

In a preferred embodiment, the first polypeptide chain comprises theamino acid sequence:

(SEQ ID NO: 57) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 56) gcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcata.

In a preferred embodiment, the second polypeptide chain comprises theamino acid sequence of SEQ ID NO:47, which is encoded by the nucleicacid sequence of SEQ ID NO:51.

As discussed above, a tetramer is provide comprising two polypeptidechains having the sequence of SEQ ID NO:57 and two polypeptide chainshaving the sequence of SEQ ID NO:47.

II.E.5 DhCpmFc(−)-G₄-GDF15(N3D):DhCpmFc(+)

The designation “DhCpmFc(−)-G₄-GDF15(N3D):DhCpmFc(+)” in the instantdisclosure refers to a heterodimer comprising (i) a first polypeptidechain comprising a GDF15(N3D) polypeptide linked to a DhCpmFc(−) domainvia a linker comprising the sequence of SEQ ID NO:58 that connects theN-terminus of the GDF15(N3D) polypeptide to the C-terminus of theDhCpmFc(−) domain and (ii) a second polypeptide chain comprising aDhCpmFc(+) domain.

In certain embodiments, a tetramer is provided, comprising a dimer oftwo DhCpmFc(−)-G₄-GDF15(N3D):DhCpmFc(+) heterodimers in which the twofirst polypeptide chains of each heterodimer are linked via aninterchain disulfide bond between their respective GDF15 regions.

More particularly, in a specific embodiment, the tetramer comprises:

(a) two DhCpmFc(+) domains (one each heterodimer) having the sequence ofSEQ ID NO:283,

(b) two DhCpmFc(+) domains (one each heterodimer) having the sequence ofSEQ ID NO:48,

(c) two a GDF15(N3D) polypeptides (one each heterodimer) having thesequence of SEQ ID NO:52, and

(d) two polypeptide linkers (one each heterodimer) comprising thesequence:

GGGG (SEQ ID NO:58)

each linking the N-terminus of a GDF15(N3D) polypeptide to theC-terminus of a DhCpmFc(−) domain via peptide bonds.

In a preferred embodiment, the first polypeptide comprises the aminoacid sequence (linker sequence double underlined):

(SEQ ID NO: 60) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGARDGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 59) gcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtggaggtggtggagcgcgcgacggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcata.

In a preferred embodiment, the second polypeptide chain comprises theamino acid sequence of SEQ ID NO:47, which is encoded by the nucleicacid sequence of SEQ ID NO:51.

As discussed above, a tetramer is provided comprising two polypeptidechains having the sequence of SEQ ID NO:60 and two polypeptide chainshaving the sequence of SEQ ID NO:47.

II.E.6 DhCpmFc(−)-G₄S-GDF15:DhCpmFc(+)

The designation “DhCpmFc(−)-G₄S-GDF15:DhCpmFc(+)” in the instantdisclosure refers to a heterodimer comprising (i) a first polypeptidechain comprising a GDF15 polypeptide linked to a DhCpmFc(−) domain via apolypeptide linker comprising the sequence of SEQ ID NO:61 that connectsthe N-terminus of the GDF15 polypeptide to the C-terminus of theDhCpmFc(−) domain and (ii) a second polypeptide chain comprising aDhCpmFc(+) domain.

In certain embodiments, a tetramer is provided, comprising a dimer oftwo DhCpmFc(−)-G₄S-GDF15:DhCpmFc(+) heterodimers in which the two firstpolypeptide chains of each heterodimer are linked via an interchaindisulfide bond between their respective GDF15 regions.

More particularly, in a specific embodiment, the tetramer comprises:

(a) two DhCpmFc(+) domains (one each heterodimer) comprising thesequence of SEQ ID NO:283,

(b) two DhCpmFc(−) domains (one each heterodimer) comprising thesequence of SEQ ID NO:48,

(c) two GDF15 polypeptides (one each heterodimer) comprising thesequence of SEQ ID NO:12, and

(d) two polypeptide linkers (one each heterodimer) comprising thesequence:

GGGGS (SEQ ID NO:61)

each linking the N-terminus of a GDF15 polypeptide to the C-terminus ofa DhCpmFc(−) domain via peptide bonds.

In a preferred embodiment, the first polypeptide comprises the aminoacid sequence (linker double underlined):

(SEQ ID NO: 63) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 62) gcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtggaggtggtggatccgcgcgcaacggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcatat ga.

In a preferred embodiment, the second polypeptide comprises the aminoacid sequence of SEQ ID NO:47, which is encoded by the nucleic acidsequence of SEQ ID NO:51.

As discussed above, a tetramer is provided comprising two polypeptidechains having the sequence of SEQ ID NO:63 and two polypeptide chainshaving the sequence of SEQ ID NO:47.

II.E.7 DhCpmFc(−)-(G₄S)₂-GDF15:DhCpmFc(+)

The designation “DhCpmFc(−)-(G₄S)₂-GDF15:DhCpmFc(+)” in the instantdisclosure refers to a heterodimer comprising (i) a first polypeptidechain comprising a GDF15 polypeptide linked to a DhCpmFc(−) domain via apolypeptide linker comprising the sequence of SEQ ID NO:64 that connectsN-terminus of the GDF15 polypeptide to the C-terminus of the DhCpmFc(−)domain and (ii) a second polypeptide chain comprising a DhCpmFc(+)domain.

In certain embodiments, a tetramer is provided, comprising a dimer oftwo DhCpmFc(−)-(G₄S)₂-GDF15:DhCpmFc(+) heterodimers in which the twofirst polypeptide chains of each heterodimer are linked via aninterchain disulfide bond between their respective GDF15 regions.

More particularly, in a specific embodiment, the tetramer comprises:

(a) two DhCpmFc(+) domains (one each heterodimer) comprising thesequence of SEQ ID NO:283,

(b) two DhCpmFc(−) domains (one each heterodimer) comprising thesequence of SEQ ID NO:48,

(c) two GDF15 polypeptides (one each heterodimer) comprising thesequence of SEQ ID NO:12, and

(d) two polypeptide linkers (one each heterodimer) comprising thesequence:

GGGGSGGGGS (SEQ ID NO:64),

each linking the N-terminus of a GDF15 polypeptide to the C-terminus ofa DhCpmFc(−) sequence via a peptide bond.

In a preferred embodiment, the first polypeptide chain comprises theamino acid sequence (linker double underlined):

(SEQ ID NO: 66) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 65) gcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtggaggtggtggatccggaggcggtggaagcgcgcgcaacggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaag actgccactgcata.

In a preferred embodiment, the second polypeptide chain comprises theamino acid sequence of SEQ ID NO:47, which is encoded by the nucleicacid sequence of SEQ ID NO:51.

As discussed above, a tetramer is provided comprising two polypeptidechains comprising the sequence of SEQ ID NO:66 and two polypeptidechains comprising the sequence of SEQ ID NO:47.

II.E.8 DhCpmFc(−)-(G₄S)₂-GDF15(N3D):DhCpmFc(+)

The designation “DhCpmFc(−)-(G₄S)₂-GDF15(N3D):DhCpmFc(+)” in the instantdisclosure refers to a heterodimer comprising (i) a first polypeptidechain comprising a GDF15(N3D) polypeptide linked to a DhCpmFc(−) domainvia a linker comprising the sequence of SEQ ID NO:64 that connects theN-terminus of the GDF15(N3D) polypeptide to the C-terminus of theDhCpmFc(−) domain and (ii) a second polypeptide chain comprising aDhCpmFc(+) domain.

In certain embodiments, a tetramer is provided, comprising a dimer oftwo DhCpmFc(−)-(G₄S)₂-GDF15(N3D):DhCpmFc(+) heterodimers in which thetwo first polypeptide chains of each heterodimer are linked via aninterchain disulfide bond between their respective GDF15 regions.

More particularly, in a specific embodiment, the tetramer comprises:

(a) two DhCpmFc(+) domains (one each heterodimer) comprising thesequence of SEQ ID NO:283,

(b) two DhCpmFc(−) domains (one each heterodimer) comprising thesequence of SEQ ID NO:48,

(c) two GDF15(N3D) polypeptides (one each heterodimer) comprising thesequence of SEQ ID NO:52, and

(d) two polypeptide linkers (one each heterodimer) comprising thesequence of SEQ ID NO:64, each linking the N-terminus of a GDF15(N3D)polypeptide to the C-terminus of a DhCpmFc(−) domain via a peptide bond.

In a preferred embodiment, the first polypeptide chain comprises theamino acid sequence (linker sequence double underlined):

(SEQ ID NO: 68) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSARDGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 67) gcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtggaggtggtggatccggaggcggtggaagcgcgcgcgacggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaag actgccactgcata.

In a preferred embodiment, the second polypeptide chain comprises theamino acid sequence of SEQ ID NO:47, which is encoded by the nucleicacid sequence of SEQ ID NO:51.

As discussed above, a tetramer is provided comprising two polypeptidechains having the sequence of SEQ ID NO:68 and two polypeptide chainshaving the sequence of SEQ ID NO:47.

II.E.9 DhCpmFc(−)-G₄P-GDF15:DhCpmFc(+)

The designation “DhCpmFc(−)-(G₄P)-GDF15:DhCpmFc(+)” in the instantdisclosure refers to a heterodimer comprising (i) a first polypeptidechain comprising a GDF15 polypeptide linked to a DhCpmFc(−) domain via alinker comprising the sequence of SEQ ID NO:69 that connects theN-terminus

of the GDF15 polypeptide to the C-terminus of the DhCpmFc(−) domain and(ii) a second polypeptide chain comprising a DhCpmFc(+) domain.

In certain embodiments, a tetramer is provided, comprising a dimer oftwo DhCpmFc(−)-(G₄P)-GDF15:DhCpmFc(+) heterodimers in which the twofirst polypeptide chains of each heterodimer are linked via aninterchain disulfide bond between their respective GDF15 regions.

More particularly, in a specific embodiment, the tetramer comprises:

(a) two DhCpmFc(+) domains (one each heterodimer) comprising thesequence of SEQ ID NO:283,

(b) two DhCpmFc(−) domains (one each heterodimer) comprising thesequence of SEQ ID NO:48,

(c) two GDF15 polypeptides (one each heterodimer) comprising thesequence of SEQ ID NO:12, and

(d) two polypeptide linkers (one each heterodimer) comprising thesequence:

GGGGP (SEQ ID NO:69),

each linking the N-terminus of a GDF15 polypeptide to the C-terminus ofa DhCpmFc(−) domain via a peptide bond.

In a preferred embodiment, the first polypeptide chain comprises theamino acid sequence (linker sequence double underlined):

(SEQ ID NO: 71) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGPARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 70) gcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtggaggtggtggacccgcgcgcaacggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcata.

In a preferred embodiment, the second polypeptide chain comprises theamino acid sequence of SEQ ID NO:47, which is encoded by the nucleicacid sequence of SEQ ID NO:51.

As discussed above, a tetramer is provided comprising two polypeptidechains having the sequence of SEQ ID NO:71 and two polypeptide chainshaving the sequence of SEQ ID NO:47.

II.E.10 DhCpmFc(−)-(G₄P)₂-GDF15:DhCpmFc(+)

The designation “DhCpmFc(−)-(G₄P)₂-GDF15:DhCpmFc(+)” in the instantdisclosure refers to a heterodimer comprising (i) a first polypeptidechain comprising a GDF15 polypeptide linked to a DhCpmFc(−) domain via alinker comprising the sequence of SEQ ID NO:72 that connects theN-terminus of the GDF15 polypeptide to the C-terminus of the DhCpmFc(−)domain and (ii) a second v comprising a DhCpmFc(+) sequence.

In certain embodiments, a tetramer is provided, comprising a dimer oftwo DhCpmFc(−)-(G₄P)₂-GDF15:DhCpmFc(+) heterodimers in which the twofirst polypeptide chains of each heterodimer are linked via aninterchain disulfide bond between their respective GDF15 regions.

More particularly, in a specific embodiment, the tetramer comprises:

(a) two DhCpmFc(+) domains (one each heterodimer) comprising thesequence of SEQ ID NO:283,

(b) two DhCpmFc(−) domains (one each heterodimer) comprising thesequence of SEQ ID NO:48,

(c) two GDF15 polypeptides (one each heterodimer) comprising thesequence of SEQ ID NO:12, and

(d) two polypeptide linkers (one each heterodimer) comprising thesequence:

GGGGPGGGGP (SEQ ID NO:72),

each linking the N-terminus of a GDF15 polypeptide to the C-terminus ofa DhCpmFc(−) domain via a peptide bond.

In a preferred embodiment, the first polypeptide chain comprises theamino acid sequence (linker sequence double underlined):

(SEQ ID NO: 74) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGPGGGGPARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 73) gcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtggaggtggtggacctggaggcggtggaccagcgcgcaacggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaag actgccactgcatatga.

In a preferred embodiment, the second polypeptide chain comprises theamino acid sequence of SEQ ID NO:47, which is encoded by the nucleicacid sequence of SEQ ID NO:51.

As discussed above, a tetramer is provided comprising two polypeptidechains having the sequence of SEQ ID NO:74 and two polypeptide chainscomprising the sequence of SEQ ID NO:47.

II.E.11 DhCpmFc(−)-G₄Q-GDF15:DhCpmFc(+)

The designation “DhCpmFc(−)-G₄Q-GDF15:DhCpmFc(+)” in the instantdisclosure refers to a heterodimer comprising (i) a first polypeptidechain comprising a GDF15 polypeptide linked to a DhCpmFc(−) domain via alinker comprising the sequence of SEQ ID NO:75 that connects theN-terminus of the GDF15 polypeptide to the C-terminus of the DhCpmFc(−)domain and (ii) a second polypeptide chain comprising a DhCpmFc(+)domain.

In certain embodiments, a tetramer is provided, comprising a dimer oftwo DhCpmFc(−)-G₄Q-GDF15:DhCpmFc(+) heterodimers in which the two firstpolypeptide chains of each heterodimer are linked via an interchaindisulfide bond between their respective GDF15 regions.

More particularly, in a specific embodiment, the tetramer comprises:

(a) two DhCpmFc(+) chains (one each heterodimer) comprising the sequenceof SEQ ID NO:283,

(b) two DhCpmFc(−) chains (one each heterodimer) comprising the sequenceof SEQ ID NO:48,

(c) two GDF15 polypeptides (one each heterodimer) comprising thesequence of SEQ ID NO:12, and

(d) two polypeptide linkers (one each heterodimer) comprising thesequence:

GGGGQ (SEQ ID NO:75)

each linking the N-terminus of a GDF15 polypeptide to the C-terminus ofa DhCpmFc(−) domain via a peptide bond.

In a preferred embodiment, the first polypeptide chain comprises theamino acid sequence (linker sequence double underlined):

(SEQ ID NO: 77) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGQARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 76) gcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcactgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtggaggtggtggacaggcgcgcaacggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcatat ga.

In a preferred embodiment, the second polypeptide chain comprises theamino acid sequence of SEQ ID NO:47, which is encoded by the nucleicacid sequence of SEQ ID NO:51.

As discussed above, a tetramer is provided comprising two polypeptidechains having the sequence of SEQ ID NO:77 and two polypeptide chainshaving the sequence of SEQ ID NO:47.

II.E.12 DhCpmFc(−)-(G₄Q)₂-GDF15:DhCpmFc(+)

The designation “DhCpmFc(−)-(G₄Q)₂-GDF15:DhCpmFc(+)” in the instantdisclosure refers to a heterodimer comprising (i) a first polypeptidechain comprising a GDF15 polypeptide linked to a DhCpmFc(−) domain via alinker comprising the sequence of SEQ ID NO:78 that connects theN-terminus of the GDF15 polypeptide to the C-terminus of the DhCpmFc(−)domain and (ii) a second polypeptide chain comprising a DhCpmFc(+)domain.

In certain embodiments, a tetramer is provided, comprising a dimer oftwo DhCpmFc(−)-(G₄Q)₂-GDF15:DhCpmFc(+) heterodimers in which the twofirst polypeptide chains of each heterodimer are linked via aninterchain disulfide bond between their respective GDF15 regions.

More particularly, in a specific embodiment, the tetramer comprises:

(a) two DhCpmFc(+) domains (one each heterodimer) comprising thesequence of SEQ ID NO:283,

(b) two DhCpmFc(−) domains (one each heterodimer) comprising thesequence of SEQ ID NO:48,

(c) two GDF15 polypeptides (one each heterodimer) comprising thesequence of SEQ ID NO:12, and

(d) two polypeptide linkers (one each heterodimer) comprising thesequence:

GGGGQGGGGQ (SEQ ID NO:78)

each linking the N-terminus of a GDF15 polypeptide to the C-terminus ofa DhCpmFc(−) domain via a peptide bond.

In a preferred embodiment, the first polypeptide chain comprises theamino acid sequence (linker sequence double underlined):

(SEQ ID NO: 80) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGQGGGGQARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 79) gcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtggaggtggtggacagggaggcggtggacaggcgcgcaacggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaag actgccactgcatatga.

In a preferred embodiment, the second polypeptide chain comprises theamino acid sequence of SEQ ID NO:47, which is encoded by the nucleicacid sequence of SEQ ID NO:51.

As discussed above, a tetramer is provided comprising two polypeptidechains having the sequence of SEQ ID NO:80 and two polypeptide chainshaving the sequence of SEQ ID NO:47.

II.E.13 DhCpmFc(−)-(G₄Q)₂-GDF15(N3D):DhCpmFc(+)

The designation “DhCpmFc(−)-(G₄Q)₂-GDF15(N3D):DhCpmFc(+)” in the instantdisclosure refers to a heterodimer comprising (i) a first polypeptidechain comprising a GDF15(N3D) polypeptide linked to a DhCpmFc(−) domainvia a linker comprising the sequence of SEQ ID NO:78 that connects theN-terminus of the GDF15(N3D) polypeptide to the C-terminus of theDhCpmFc(−) domain and (ii) a second polypeptide chain comprising aDhCpmFc(+) domain.

In certain embodiments, a tetramer is provided, comprising a dimer oftwo DhCpmFc(−)-(G₄Q)₂-GDF15(N3D):DhCpmFc(+) heterodimers in which thetwo first polypeptide chains of each heterodimer are linked via aninterchain disulfide bond between their respective GDF15 regions.

More particularly, in a specific embodiment, the tetramer comprises:

(a) two DhCpmFc(+) domains (one each heterodimer) comprising thesequence of SEQ ID NO:283,

(b) two DhCpmFc(−) domains (one each heterodimer) comprising thesequence of SEQ ID NO:48,

(c) two GDF15(N3D) polypeptides (one each heterodimer) comprising thesequence of SEQ ID NO:52, and

(d) two polypeptide linkers (one each heterodimer) comprising thesequence of SEQ ID NO:78 each linking the N-terminus of a GDF15(N3D)polypeptide to the C-terminus of a DhCpmFc(−) domain via a peptide bond.

In a preferred embodiment, the first polypeptide chain comprises theamino acid sequence (linker sequence double underlined):

(SEQ ID NO: 82) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGQGGGGQARDGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 81) gcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtggaggtggtggacagggaggcggtggacaggcgcgcgacggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaag actgccactgcata.

In a preferred embodiment, the second polypeptide chain comprises theamino acid sequence of SEQ ID NO:47, which is encoded by the nucleicacid sequence of SEQ ID NO:51.

As discussed above, a tetramer is provided comprising two polypeptidechains having the sequence of SEQ ID NO:82 and two polypeptide chainshaving the sequence of SEQ ID NO:47.

II.E.14 DhCpmFc(−)-(G₄Q)₂-GDF15(Ndel3):DhCpmFc(+)

The designation “DhCpmFc(−)-(G₄Q)₂-GDF15(Ndel3):DhCpmFc(+)” in theinstant disclosure refers to a heterodimer comprising (i) a firstpolypeptide chain comprising a GDF15(Ndel3) polypeptide linked to aDhCpmFc(−) domain via a linker comprising the sequence of SEQ ID NO:78that connects the N-terminus of the GDF15(Ndel3) polypeptide to theC-terminus of the DhCpmFc(−) domain and (ii) a second polypeptide chaincomprising a DhCpmFc(+) domain.

In certain embodiments, a tetramer is provided, comprising a dimer oftwo DhCpmFc(−)-(G₄Q)₂-GDF15(Ndel3):DhCpmFc(+) heterodimers in which thetwo first polypeptide chains of each heterodimer are linked via aninterchain disulfide bond between their respective GDF15 regions.

More particularly, in a specific embodiment, the tetramer comprises:

(a) two DhCpmFc(+) domains (one each heterodimer) comprising thesequence of SEQ ID NO:283,

(b) two DhCpmFc(−) domains (one each heterodimer) comprising thesequence of SEQ ID NO:48,

(c) two GDF15(Ndel3) polypeptides (one each heterodimer) comprising thesequence of SEQ ID NO:55, and

(d) two polypeptide linkers (one each heterodimer) comprising thesequence of SEQ ID NO:78 each linking the N-terminus of a GDF15(Ndel3)polypeptide to the C-terminus of a DhCpmFc(−) domain via a peptide bond.

In a preferred embodiment, the first polypeptide chain comprises theamino acid sequence (linker sequence double underlined):

(SEQ ID NO: 84) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGQGGGGQGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 83) gcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtggaggtggtggacagggaggcggtggacagggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccact gcata.

In a preferred embodiment, the second polypeptide chain comprises theamino acid sequence of SEQ ID NO:47, which is encoded by the nucleicacid sequence of SEQ ID NO:51.

As discussed above, a tetramer is provided comprising two polypeptidechains having the sequence of SEQ ID NO:84 and two polypeptide chainshaving the sequence of SEQ ID NO:47.

II.F. Charged Pair (delHinge) Cysteine Clamp

A “cysteine clamp” mutation may be introduced into a Fc domain, such asa CpmFc(+) domain, a CpmFc(−) domain, a DhCpmFc(+) domain, or aDhCpmFc(−) domain. A “cysteine clamp” mutation typically involves theintroduction of a cysteine into the CH3 domain of an Fc domain at aspecific location through mutation so that when incubated with anotherFc domain, also having a cysteine introduced into the CH3 domain at aspecific location through mutation, a disulfide bond (cysteine clamp)may be formed between the two Fc domains (e.g., between a CpmFc(+)domain having a “cysteine clamp” mutation and a CpmFc(−) domain having a“cysteine clamp” mutation or between a DhCpmFc(+) domain having a“cysteine clamp” mutation and a DhCpmFc(−) domain having a “cysteineclamp” mutation). An Fc domain may contain one or more such cysteineclamp mutations.

In one embodiment, a cysteine clamp is provided by introducing a serineto cysteine mutation (S354C) into a first Fc domain and a tyrosine tocysteine mutation (Y349C) into a second Fc domain.

The designation “DhCpmFc(−)(S354C)” domain in the instant disclosurerefers to an DhCpmFc(−) domain comprising a serine to cysteine mutation(S354C). The designation “DhCpmFc(+)(S354C)” domain in the instantdisclosure refers to an DhCpmFc(+) domain comprising a serine tocysteine mutation (S354C). The designation “DhCpmFc(−)(Y349C)” domain inthe instant disclosure refers to an DhCpmFc(−) domain comprising aserine to cysteine mutation (Y349C). The designation “DhCpmFc(+)(Y349C)”domain in the instant disclosure refers to an DhCpmFc(+) domaincomprising a serine to cysteine mutation (Y349C).

The designation “CpmFc(−)(S354C)” domain in the instant disclosurerefers to an CpmFc(−) domain comprising a serine to cysteine mutation(S354C). The designation “CpmFc(+)(S354C)” domain in the instantdisclosure refers to an CpmFc(+) domain comprising a serine to cysteinemutation (S354C). The designation “CpmFc(−)(Y349C)” domain in theinstant disclosure refers to an CpmFc(−) domain comprising a serine tocysteine mutation (Y349C). The designation “CpmFc(+)(Y349C)” domain inthe instant disclosure refers to an CpmFc(+) domain comprising a serineto cysteine mutation (Y349C).

In another embodiment, a cysteine clamp is provided by introducing aleucine to cysteine mutation (L351C) into both a first and Fc domain.

The designation “DhCpmFc(−)(L351C)” domain in the instant disclosurerefers to an DhCpmFc(−) domain comprising a serine to cysteine mutation(L351C). The designation “DhCpmFc(+)(L351C)” domain in the instantdisclosure refers to an DhCpmFc(+) domain comprising a serine tocysteine mutation (L351C).

The designation “CpmFc(−)(L351C)” domain in the instant disclosurerefers to an CpmFc(−) domain comprising a serine to cysteine mutation(L351C). The designation “CpmFc(+)(L351C)” domain in the instantdisclosure refers to an CpmFc(+) domain comprising a serine to cysteinemutation (L351C).

The C-terminal lysine (K447) optionally may be deleted in the CpmFc(+)domain, the CpmFc(−) domain, or both. This may be advantageous, forexample, when a peptide is fused at the C-terminus to reduce proteolysisof the fusion protein.

In some embodiments a heterodimer is provided comprises (i) a firstpolypeptide chain comprising a GDF15 region linked to a CpmFc(+) domaincomprising a cysteine clamp mutation and (ii) a second polypeptide chaincomprising a CpmFc(−) domain comprising a cysteine clamp mutation. Insome embodiments, the N-terminus of the GDF15 region is linked to theC-terminus of the CpmFc(+) domain comprising a cysteine clamp mutation,directly or via a polypeptide linker. In other embodiments, theN-terminus of the CpmFc(+) domain comprising a cysteine clamp mutationis linked to the C-terminus of the GDF15 region, directly or via apolypeptide linker.

In some embodiments a heterodimer is provided comprises (i) a firstpolypeptide chain comprising a GDF15 region linked to a CpmFc(−) domaincomprising a cysteine clamp mutation and (ii) a second polypeptide chaincomprising a CpmFc(+) domain comprising a cysteine clamp mutation. Insome embodiments, the N-terminus of the GDF15 region is linked to theC-terminus of the CpmFc(−) domain comprising a cysteine clamp mutation,directly or via a polypeptide linker. In other embodiments, theN-terminus of the CpmFc(−) domain comprising a cysteine clamp mutationis linked to the C-terminus of the GDF15 region, directly or via apolypeptide linker.

In some embodiments a heterodimer is provided comprises (i) a firstpolypeptide chain comprising a GDF15 region linked to a DhCpmFc(+)domain comprising a cysteine clamp mutation and (ii) a secondpolypeptide chain comprising a DhCpmFc(−) domain comprising a cysteineclamp mutation. In some embodiments, the N-terminus of the GDF15 regionis linked to the C-terminus of the DhCpmFc(+) domain comprising acysteine clamp mutation, directly or via a polypeptide linker. In otherembodiments, the N-terminus of the DhCpmFc(+) domain comprising acysteine clamp mutation is linked to the C-terminus of the GDF15 region,directly or via a polypeptide linker.

In some embodiments a heterodimer is provided comprises (i) a firstpolypeptide chain comprising a GDF15 region linked to a DhCpmFc(−)domain comprising a cysteine clamp mutation and (ii) a secondpolypeptide chain comprising a DhCpmFc(+) domain comprising a cysteineclamp mutation. In some embodiments, the N-terminus of the GDF15 regionis linked to the C-terminus of the DhCpmFc(−) domain comprising acysteine clamp mutation, directly or via a polypeptide linker. In otherembodiments, the N-terminus of the DhCpmFc(−) domain comprising acysteine clamp mutation is linked to the C-terminus of the GDF15 region,directly or via a polypeptide linker.

In some embodiments, a tetramer is provided comprising a dimer of twosuch heterodimers in which the two first polypeptide chains of theheterodimers are linked via an interchain disulfide bond between theirrespective GDF15 regions. See FIG. 5 for a graphic depiction of anembodiment of a tetramer comprising two heterodimers, where eachheterodimer comprises (i) a first polypeptide chain comprising a GDF15polypeptide linked via a polypeptide linker to a DhCpmFc(+)(L351C)domain and (ii) a second polypeptide chain comprising aDhCpmFc(−)(L351C) domain.

II.F.1 DhCpmFc(+)(S354C)-GDF15(N3D):DhCpmFc(−)(Y349C)

The designation “DhCpmFc(+)(S354C)-GDF15(N3D):DhCpmFc(−)(Y349C)” in theinstant disclosure refers to a heterodimer comprising (i) a firstpolypeptide chain comprising a GDF15(N3D) polypeptide, the N-terminus ofwhich is linked directly to the C-terminus of a DhCpmFc(+)(S354C)domain, and (ii) a second polypeptide chain comprising aDhCpmFc(−)(Y349C) domain. The cysteine clamp mutations allow the firstand second polypeptide chains to be linked via an interchain disulfidebond between C354 of the first polypeptide chain and C349 of the secondpolypeptide chain.

In certain embodiments, a tetramer is provided, comprising a dimer oftwo DhCpmFc(+)(S354C)-GDF15(N3D):DhCpmFc(−)(Y349C) heterodimers in whichthe two first polypeptide chains of each heterodimer are linked via aninterchain disulfide bond between their respective GDF15 regions.

More particularly, in a specific embodiment, the tetramer comprises:

(a) two DhCpmFc(+)(S354C) domains (one each heterodimer) comprising thesequence:

(SEQ ID NO: 85) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPG,

(b) two DhCpmFc(−)(Y349C) domains (one each heterodimer) comprising thesequence:

(SEQ ID NO: 284) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPG,and

(c) two GDF15(N3D) polypeptides (one each heterodimer) comprising thesequence of SEQ ID NO:52.

In a preferred embodiment, the first polypeptide chain comprises theamino acid sequence:

(SEQ ID NO: 88) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGARDGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 87) gccccagagctgcttggtggaccatccgtgttcctgtttcctccaaagccgaaggacaccctgatgatctcaagaactccggaagtgacttgcgtcgtcgtggacgtgtcacatgaggatccagaggtcaagttcaattggtatgtggacggagtggaagtgcataacgccaagaccaaaccccgcgaagaacagtacaatagcacctaccgcgtggtgagcgtccttactgtgctccaccaggactggcttaatgggaaggaatacaagtgtaaggtgtccaacaaggccctccccgctcccatcgaaaagaccatctcaaaggcaaaggggcaaccaagggaacctcaagtgtacaccctgcctccgtgcaggaaggagatgaccaagaaccaggtcagcctgacttgtctcgtgaagggcttctatcccagcgatattgctgtggaatgggagtcaaatggccagcccgagaataactacaaaactaccccacccgtgctgaaatctgatgggtccttcttcctttactccaagctgaccgtggacaagagccgctggcaacaaggcaatgtctttagctgctcagtgatgcatgaggctctccataatcactacactcagaagtcactgtccctgtcacctggcgcgcgcgacggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcata.

In a preferred embodiment, the second polypeptide chain and comprisesthe amino acid sequence:

(SEQ ID NO: 86) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 89) gcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtgcaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaa a.

As discussed above, a tetramer is provided comprising two polypeptidechains comprising the sequence of SEQ ID NO:88 and two polypeptidechains comprising the sequence of SEQ ID NO:86.

II.F.2 DhCpmFc(−)(Y349C)-GDF15:DhCpmFc(+)(S354C)

The designation “DhCpmFc(−)(Y349C)-GDF15:DhCpmFc(+)(S354C)” in theinstant disclosure refers to a heterodimer comprising (i) a firstpolypeptide chain comprising a GDF15 polypeptide, the N-terminus ofwhich is linked directly to the C-terminus of a DhCpmFc(−)(Y349C)domain, and (ii) a second polypeptide chain comprising aDhCpmFc(+)(S345C) domain. The cysteine clamp mutations allow the firstand second polypeptide chains to be linked via an interchain disulfidebond between C349 of the first polypeptide chain and C354 of the secondpolypeptide chain.

In certain embodiments, a tetramer is provided, comprising a dimer oftwo DhCpmFc(−)(Y349C)-GDF15:DhCpmFc(+)(S354C) heterodimers in which thetwo first polypeptide chains of each heterodimer are linked via aninterchain disulfide bond between their respective GDF15 regions.

More particularly, in a specific embodiment, the tetramer comprises:

(a) two DhCpmFc(+)(S354C) domains (one each heterodimer) comprising thesequence:

(SEQ ID NO: 285) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPG,

(b) two DhCpmFc(−)(Y349C) domains (one each heterodimer) comprising thesequence:

(SEQ ID NO: 91) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPG,and

(c) two GDF15 polypeptides (one each heterodimer) comprising thesequence of SEQ ID NO:12.

In a preferred embodiment, the first polypeptide chain comprises theamino acid sequence:

(SEQ ID NO: 93) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 92) gcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtgcaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtgcgcgcaacggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcata.

In a preferred embodiment, the second polypeptide chain comprises thesequence:

(SEQ ID NO: 90) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 94) gccccagagctgcttggtggaccatccgtgttcctgtttcctccaaagccgaaggacaccctgatgatctcaagaactccggaagtgacttgcgtcgtcgtggacgtgtcacatgaggatccagaggtcaagttcaattggtatgtggacggagtggaagtgcataacgccaagaccaaaccccgcgaagaacagtacaatagcacctaccgcgtggtgagcgtccttactgtgctccaccaggactggcttaatgggaaggaatacaagtgtaaggtgtccaacaaggccctccccgctcccatcgaaaagaccatctcaaaggcaaaggggcaaccaagggaacctcaagtgtacaccctgcctccgtgcaggaaggagatgaccaagaaccaggtcagcctgacttgtctcgtgaagggcttctatcccagcgatattgctgtggaatgggagtcaaatggccagcccgagaataactacaaaactaccccacccgtgctgaaatctgatgggtccttcttcctttactccaagctgaccgtggacaagagccgctggcaacaaggcaatgtctttagctgctcagtgatgcatgaggctctccataatcactacactcagaagtcactgtccctgtctccgggtaa a.

As discussed above, a tetramer is provided comprising two polypeptidechains comprising the sequence of SEQ ID NO:93 and two polypeptidechains comprising the sequence of SEQ ID NO:90.

II.F.3 DhCpmFc(−)(Y349C)-GDF15(N3D):DhCpmFc(+)(S354C)

The designation “DhCpmFc(−)(Y349C)-GDF15(N3D):DhCpmFc(+)(S354C)” in theinstant disclosure refers to heterodimer comprising (i) a firstpolypeptide chain comprising a GDF15(N3D) polypeptide, the N-terminus ofwhich is linked directly to the C-terminus of a DhCpmFc(−)(Y349C) domainand (ii) a second polypeptide chain comprising a DhCpmFc(+)(S345C)domain. The cysteine clamp mutations allow the first and secondpolypeptide chains to be linked via an interchain disulfide bond betweenC349 of the first polypeptide and C354 of the second polypeptide.

In certain embodiments, a tetramer is provided, comprising a dimer oftwo DhCpmFc(−)(Y349C)-GDF15(N3D):DhCpmFc(+)(S354C) heterodimers in whichthe two first polypeptide chains of each heterodimer are linked via aninterchain disulfide bond between their respective GDF15 regions. An N3Dmutation in GDF15 sequence may be introduced, e.g., to eliminateheterogeneity caused by N deamidation.

More particularly, in a specific embodiment, the tetramer comprises:

(a) two DhCpmFc(+)(S354C) domains (one each heterodimer) comprising thesequence of SEQ ID NO:285,

(b) two DhCpmFc(−)(Y349C) domains (one each heterodimer) comprising thesequence of SEQ ID NO:91, and

(c) two GDF15(N3D) polypeptides (one each heterodimer) comprising thesequence of SEQ ID NO:52.

In a preferred embodiment, the first polypeptide chain comprises theamino acid sequence:

(SEQ ID NO: 96) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGARDGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 95) gcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtgcaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtgcgcgcgacggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcata.

In a preferred embodiment, the second polypeptide chain comprises theamino acid sequence of SEQ ID NO:90, which is encoded by the nucleicacid sequence of SEQ ID NO:94.

As discussed above, a tetramer is provided comprising two polypeptidechains comprising the sequence of SEQ ID NO:96 and two polypeptidechains comprising the sequence of SEQ ID NO:90.

II.F.4 DhCpmFc(−)(Y349C)-GDF15(Ndel3):DhCpmFc(+)(S354C)

The designation “DhCpmFc(−)(Y349C)-GDF15(Ndel3):DhCpmFc(+)(S354C)” inthe instant disclosure refers to a heterodimer comprising (i) a firstpolypeptide chain comprising a GDF15(Ndel3) polypeptide, the N-terminusof which is linked directly to the C-terminus of a DhCpmFc(−)(Y349C)domain, and (ii) a second polypeptide chain comprising aDhCpmFc(+)(S345C) domain. The cysteine clamp mutations allow the firstand second polypeptide chains to be linked via an interchain disulfidebond between C349 of the first polypeptide chain and C354 of the secondpolypeptide chain.

In certain embodiments, a tetramer is provided, comprising a dimer oftwo DhCpmFc(−)(Y349C)-GDF15(Ndel3):DhCpmFc(+)(S354C) heterodimers inwhich the two first polypeptide chains of each heterodimer are linkedvia an interchain disulfide bond between their respective GDF15 regions.Deletion of the N-terminal 3 amino acids (Ndel3) in GDF15 sequence maybe introduced, e.g., to eliminate heterogeneity caused by deamidation ofasparagines or isomerization of aspartic acid.

More particularly, in a specific embodiment, the tetramer comprises:

(a) two DhCpmFc(+)(S354C) domains (one each heterodimer) of comprisingthe sequence of SEQ ID NO:285,

(b) two DhCpmFc(−)(Y349C) domains (one each heterodimer) comprising thesequence of SEQ ID NO:91, and

(c) two GDF15(Ndel3) polypeptides (one each heterodimer) comprising thesequence of SEQ ID NO:55.

In a preferred embodiment, the first polypeptide chain comprises theamino acid sequence:

(SEQ ID NO: 98) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 97) gcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtgcaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcata.

In a preferred embodiment, the second polypeptide chain comprises theamino acid sequence of SEQ ID NO:90, which is encoded by the nucleicacid sequence of SEQ ID NO:94.

As discussed above, a tetramer is provided comprising two polypeptidechains comprising the sequence of SEQ ID NO:98 and two polypeptidechains comprising the sequence of SEQ ID NO:90.

II.F.5 DhCpmFc(−)(Y349C)-G₄-GDF15(N3D):DhCpmFc(+)(S354C)

The designation “DhCpmFc(−)(Y349C)-G₄-GDF15(N3D):DhCpmFc(+)(S354C)” inthe instant disclosure refers to a heterodimer comprising (i) a firstpolypeptide chain comprising a GDF15(N3D) polypeptide linked to aDhCpmFc(−)(Y349C) domain via a linker comprising the sequence of SEQ IDNO:58 that connects the N-terminus of the GDF15(N3D) polypeptide to theC-terminus of a DhCpmFc(−)(Y349C) domain and (ii) a second polypeptidechain comprising a DhCpmFc(+)(S345C) domain. The cysteine clampmutations allow the first and second polypeptide chains to be linked viaan interchain disulfide bond between C349 of the first polypeptide chainand C354 of the second polypeptide chain.

In certain embodiments, a tetramer is provided, comprising a dimer oftwo DhCpmFc(−)(Y349C)-G₄-GDF15(N3D):DhCpmFc(+)(S354C) heterodimers inwhich the two first polypeptide chains of each heterodimer are linkedvia an interchain disulfide bond between their respective GDF15 regions.

More particularly, in a specific embodiment, the tetramer comprises:

(a) two DhCpmFc(+)(S354C) domains (one each heterodimer) of comprisingthe sequence of SEQ ID NO:285,

(b) two DhCpmFc(−)(Y349C) domains (one each heterodimer) comprising thesequence of SEQ ID NO:91,

(c) two GDF15(N3D) polypeptides (one each heterodimer) comprising thesequence of SEQ ID NO:52, and

(d) two polypeptide linkers (one each heterodimer) comprising thesequence of SEQ ID NO:58 each linking the N-terminus of a GDF15(N3D)polypeptide to the C-terminus of a DhCpmFc(−)(Y349C) domain via apeptide bond.

In a preferred embodiment, the first polypeptide chain comprises theamino acid sequence (linker sequence double underlined):

(SEQ ID NO: 100) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGARDGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 99) gcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtgcaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtggtggaggtggtgcgcgcgacggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcata.

In a preferred embodiment, the second polypeptide chain comprises theamino acid sequence of SEQ ID NO:90, which is encoded by the nucleicacid sequence of SEQ ID NO:94.

As discussed above, in a specific embodiment, a heterotetramer isprovided comprising two monomers having the sequence of SEQ ID NO:100and two monomers having the sequence of SEQ ID NO:90.

II.F.6 DhCpmFc(−)(Y349C)-(G₄S)₂-GDF15(N3D):DhCpmFc(+)(S354C)

The designation “DhCpmFc(−)(Y349C)-(G₄S)₂-GDF15(N3D):DhCpmFc(+)(S354C)”in the instant disclosure refers to a heterodimer comprising (i) a firstpolypeptide chain comprising a GDF15(N3D) polypeptide linked to aDhCpmFc(−)(Y349C) domain via a linker comprising the sequence of SEQ IDNO:64 that connects the N-terminus of the GDF15(N3D) polypeptide to theC-terminus of a DhCpmFc(−)(Y349C) domain, and (ii) a second polypeptidechain comprising a DhCpmFc(+)(S345C) domain. The cysteine clampmutations allow the first and second polypeptide chains to be linked viaan interchain disulfide bond between C349 of the first polypeptide chainand C354 of the second polypeptide chain.

In certain embodiments, a tetramer is provided, comprising a dimer oftwo DhCpmFc(−)(Y349C)-(G₄S)₂-GDF15(N3D):DhCpmFc(+)(S354C) heterodimersin which the two first polypeptide chains of each heterodimer are linkedvia an interchain disulfide bond between their respective GDF15 regions.

More particularly, in a specific embodiment, the tetramer:

(a) two DhCpmFc(+)(S354C) domains (one each heterodimer) of comprisingthe sequence of SEQ ID NO:285,

(b) two DhCpmFc(−)(Y349C) domains (one each heterodimer) comprising thesequence of SEQ ID NO:91,

(c) two GDF15(N3D) polypeptides (one each heterodimer) comprising thesequence of SEQ ID NO:52, and

(d) two polypeptide linkers (one each heterodimer) comprising thesequence of SEQ ID NO:64 each linking the N-terminus of a GDF15(N3D)polypeptide to the C-terminus of a DhCpmFc(−)(Y349C) domain via apeptide bond.

In a preferred embodiment, the first polypeptide comprises the aminoacid sequence (linker sequence double underlined):

(SEQ ID NO: 102) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSARDGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 101) gcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtgcaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtggaggtggtggatccggaggcggtggaagcgcgcgcgacggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaag actgccactgcata.

In a preferred embodiment, the second polypeptide chain comprises theamino acid sequence of SEQ ID NO:90, which is encoded by the nucleicacid sequence of SEQ ID NO:94.

As discussed above, in a specific embodiment, a heterotetramer isprovided comprising two monomers having the sequence of SEQ ID NO:102and two monomers having the sequence of SEQ ID NO:90.

II.F.7 DhCpmFc(−)(Y349C)-(G₄Q)₂-GDF15(N3D):DhCpmFc(+)(S354C)

The designation “DhCpmFc(−)(Y349C)-(G₄Q)₂-GDF15(N3D):DhCpmFc(+)(S354C)”in the instant disclosure refers to a heterodimer comprising (i) a firstpolypeptide chain comprising a GDF15(N3D) polypeptide linked to aDhCpmFc(−)(Y349C) domain via a linker comprising the sequence of SEQ IDNO:78 that connects the N-terminus of the GDF15(N3D) polypeptide to theC-terminus of the DhCpmFc(−)(Y349C) domain, and (ii) a secondpolypeptide chain comprising a DhCpmFc(+)(S345C) domain. The cysteineclamp mutations allow the first and second polypeptide chains to belinked via an interchain disulfide bond between C349 of the firstpolypeptide chain and C354 of the second polypeptide chain.

In certain embodiments, a tetramer is provided, comprising a dimer oftwo DhCpmFc(−)(Y349C)-(G₄Q)₂-GDF15(N3D):DhCpmFc(+)(S354C) heterodimersin which the two first polypeptide chains of each heterodimer are linkedvia an interchain disulfide bond between their respective GDF15 regions.

More particularly, in a specific embodiment, the tetramer comprises:

(a) two DhCpmFc(+)(S354C) domains (one each heterodimer) of comprisingthe sequence of SEQ ID NO:285,

(b) two DhCpmFc(−)(Y349C) domains (one each heterodimer) comprising thesequence of SEQ ID NO:91,

(c) two GDF15(N3D) polypeptides (one each heterodimer) comprising thesequence of SEQ ID NO:52, and

(d) two polypeptide linkers (one each heterodimer) comprising thesequence of SEQ ID NO:78 each linking the N-terminus of a GDF15(N3D)polypeptide to the C-terminus of a DhCpmFc(−)(Y349C) domain via apeptide bond.

In a preferred embodiment, the first polypeptide comprises the aminoacid sequence (linker sequence double underlined):

(SEQ ID NO: 104) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGQGGGGQARDGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 103) gcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtgcaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtggaggtggtggacagggaggcggtggacaggcgcgcgacggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaag actgccactgcata.

In a preferred embodiment, the second polypeptide chain comprises theamino acid sequence of SEQ ID NO:90, which is encoded by the nucleicacid sequence of SEQ ID NO:94.

As discussed above, in a specific embodiment, a heterotetramer isprovided comprising two monomers having the sequence of SEQ ID NO:104and two monomers having the sequence of SEQ ID NO:90.

II.F.8. DhCpmFc(−)(L351C)-(G₄S)₂-GDF15:DhCpmFc(+)(L351C)

The designation “DhCpmFc(−)(L351C)-(G₄S)₂-GDF15:DhCpmFc(+)(L351C)” inthe instant disclosure refers to a heterodimer comprising (i) a firstpolypeptide chain comprising a GDF15 polypeptide linked to aDhCpmFc(−)(L351C) domain via a linker comprising the sequence of SEQ IDNO:64 that connects the N-terminus of the GDF15 polypeptide to theC-terminus of the DhCpmFc(−)(L351C) domain, and (ii) a secondpolypeptide chain comprising a DhCpmFc(+)(L351C) domain. The cysteineclamp mutations allow the first and second polypeptide chains to belinked via an interchain disulfide bond between C351 of the firstpolypeptide chain and C351 of the second polypeptide chain.

In certain embodiments, a tetramer is provided, comprising a dimer oftwo DhCpmFc(−)(L351C)-(G₄S)₂-GDF15:DhCpmFc(+)(L 351C) heterodimers inwhich the two first polypeptide chains of each heterodimer are linkedvia an interchain disulfide bond between their respective GDF15 regions.

More particularly, in a specific embodiment, the tetramer comprises:

(a) two DhCpmFc(+)(L351C) domains (one each heterodimer) comprising thesequence:

(SEQ ID NO: 286) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTCPPSRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPG,

(b) two DhCpmFc(−)(L351C) domains (one each heterodimer) comprising thesequence:

(SEQ ID NO: 106) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTCPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPG,

(c) two GDF15 polypeptide chains (one each heterodimer) comprising thesequence of SEQ ID NO:12, and

(d) two polypeptide linkers (one each heterodimer) comprising thesequence of SEQ ID NO:64 each linking the N-terminus of a GDF15polypeptide to the C-terminus of a DhCpmFc(−)(L351C) domain via apeptide bond.

In a preferred embodiment, the first polypeptide chain comprises theamino acid sequence (linker sequence double underlined):

(SEQ ID NO: 108) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTCPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 107) gcgccggaactgctgggcggcccgagcgtgtttctgtttccgccgaaaccgaaagataccctgatgattagccgcaccccggaagtgacctgcgtggtggtggatgtgagccatgaagatccggaagtgaaatttaactggtatgtggatggcgtggaagtgcataacgcgaaaaccaaaccgcgcgaagaacagtataacagcacctatcgcgtggtgagcgtgctgaccgtgctgcatcaggattggctgaacggcaaagaatataaatgcaaagtgagcaacaaagcgctgccggcgccgattgaaaaaaccattagcaaagcgaaaggccagccgcgcgaaccgcaggtgtatacctgcccgccgagccgcgaagaaatgaccaaaaaccaggtgagcctgacctgcctggtgaaaggcttttatccgagcgatattgcggtggaatgggaaagcaacggccagccggaaaacaactatgataccaccccgccggtgctggatagcgatggcagcttttttctgtatagcgatctgaccgtggataaaagccgctggcagcagggcaacgtgtttagctgcagcgtgatgcatgaagcgctgcataaccattatacccagaaaagcctgagcctgagcccgggcggcggcggcggcagcggcggcggcggcagcgcgcgcaacggcgatcattgcccgctgggcccgggccgctgctgccgcctgcataccgtgcgcgcgagcctggaagatctgggctgggcggattgggtgctgagcccgcgcgaagtgcaggtgaccatgtgcattggcgcgtgcccgagccagtttcgcgcggcgaacatgcatgcgcagattaaaaccagcctgcatcgcctgaaaccggataccgtgccggcgccgtgctgcgtgccggcgagctataacccgatggtgctgattcagaaaaccgataccggcgtgagcctgcagacctatgatgatctgctggcgaaag attgccattgcatt.

In a preferred embodiment, the second polypeptide chain and comprisesthe amino acid sequence:

(SEQ ID NO: 105) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTCPPSRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 109) gcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacacctgtcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaa a.

As discussed above, in a specific embodiment, a heterotetramer isprovided comprising two monomers having the sequence of SEQ ID NO:108and two monomers having the sequence of SEQ ID NO:105.

II.G. HSA

The designations “HSA” or “human serum albumin” in the instantdisclosure refer to a fusion protein comprising a GDF15 region linked,directly or via a polypeptide linker, to a human serum albumin (HSA)polypeptide. In some embodiments, the fusion protein comprises two ormore HSA polypeptides.

Typically, the N-terminus of the GDF15 region is linked, directly or viaa polypeptide linker, to the C-terminus of the HSA polypeptide. However,in some embodiments, the N-terminus of the HSA polypeptide is linked,directly or via a polypeptide linker, to the C-terminus of the GDF15region.

In certain embodiments, a homodimer is provided comprising two suchfusion proteins linked via an interchain disulfide bond between theirrespective GDF15 regions. See FIG. 6 for a graphic depiction of anembodiment of such a homodimer. Alternatively, a heterodimer is providedcomprising one such fusion protein and a GDF15 polypeptide or GDF mutantpolypeptide, linked via an interchain disulfide bond between the GDF15region of the fusion protein and the GDF15 polypeptide or mutantpolypeptide.

II.G.1 HSA-(G₄S)₄-GDF15:GDF15 Heterodimer

The designation “HSA-(G₄S)₄-GDF15:GDF15” in the instant disclosurerefers to a heterodimer comprising (i) a first polypeptide chaincomprising a GDF15 polypeptide linked to an HSA polypeptide via a linkercomprising the sequence of SEQ ID NO:18 that connects the N-terminus ofthe GDF15 polypeptide to the C-terminus of the HSA polypeptide, and (ii)a second polypeptide chain comprising a GDF15 polypeptide.

Typically, the first and second polypeptide chains are linked via aninterchain disulfide bond between their respective GDF15 regions.

More particularly, in a specific embodiment, the heterodimer comprises:

(a) one HSA polypeptide (first monomer) comprising the sequence:

(SEQ ID NO: 110) DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL,and

(b) two GDF15 polypeptides (one each monomer) comprising the sequence ofSEQ ID NO:12, and

(c) one polypeptide linker (first monomer) comprising the sequence ofSEQ ID NO:18 linking the N-terminus of a GDF15 polypeptide to theC-terminus of the HSA polypeptide via a peptide bond.

In a preferred embodiment, the first polypeptide comprises the aminoacid sequence (linker double underlined):

(SEQ ID NO: 112) DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGGGGSGGGGSGGGGSGGGGSARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTG VSLQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 111) gatgcacacaagagtgaggttgctcatcgatttaaagatttgggagaagaaaatttcaaagccttggtgttgattgcctttgctcagtatcttcagcagtgtccatttgaagatcatgtaaaattagtgaatgaagtaactgaatttgcaaaaacatgtgttgctgatgagtcagctgaaaattgtgacaaatcacttcataccctttttggagacaaattatgcacagttgcaactcttcgtgaaacctatggtgaaatggctgactgctgtgcaaaacaagaacctgagagaaatgaatgcttcttgcaacacaaagatgacaacccaaacctcccccgattggtgagaccagaggttgatgtgatgtgcactgcttttcatgacaatgaagagacatttttgaaaaaatacttatatgaaattgccagaagacatccttacttttatgccccggaactccttttctttgctaaaaggtataaagctgcttttacagaatgttgccaagctgctgataaagctgcctgcctgttgccaaagctcgatgaacttcgggatgaagggaaggcttcgtctgccaaacagagactcaagtgtgccagtctccaaaaatttggagaaagagctttcaaagcatgggcagtagctcgcctgagccagagatttcccaaagctgagtttgcagaagtttccaagttagtgacagatcttaccaaagtccacacggaatgctgccatggagatctgcttgaatgtgctgatgacagggcggaccttgccaagtatatctgtgaaaatcaagattcgatctccagtaaactgaaggaatgctgtgaaaaacctctgttggaaaaatcccactgcattgccgaagtggaaaatgatgagatgcctgctgacttgccttcattagctgctgattttgttgaaagtaaggatgtttgcaaaaactatgctgaggcaaaggatgtcttcctgggcatgtttttgtatgaatatgcaagaaggcatcctgattactctgtcgtgctgctgctgagacttgccaagacatatgaaaccactctagagaagtgctgtgccgctgcagatcctcatgaatgctatgccaaagtgttcgatgaatttaaacctcttgtggaagagcctcagaatttaatcaaacaaaattgtgagctttttgagcagcttggagagtacaaattccagaatgcgctattagttcgttacaccaagaaagtaccccaagtgtcaactccaactcttgtagaggtctcaagaaacctaggaaaagtgggcagcaaatgttgtaaacatcctgaagcaaaaagaatgccctgtgcagaagactatctatccgtggtcctgaaccagttatgtgtgttgcatgagaaaacgccagtaagtgacagagtcaccaaatgctgcacagaatccttggtgaacaggcgaccatgcttttcagctctggaagtcgatgaaacatacgttcccaaagagtttaatgctgaaacattcaccttccatgcagatatatgcacactttctgagaaggagagacaaatcaagaaacaaactgcacttgttgagctcgtgaaacacaagcccaaggcaacaaaagagcaactgaaagctgttatggatgatttcgcagcttttgtagagaagtgctgcaaggctgacgataaggagacctgctttgccgaggagggtaaaaaacttgttgcggccagtcaggccgccttaggcttaggaggtggtggatccggaggcggtggaagcggaggtggtggatctggaggcggtggaagcgcgcgcaacggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcat atga.

In a preferred embodiment, the second polypeptide chain comprises theamino acid sequence of SEQ ID NO:12, which is encoded by the nucleicacid sequence of SEQ ID NO:11.

As discussed above, a heterodimer is provided comprising a firstpolypeptide chain having the sequence of SEQ ID NO:112 and a secondpolypeptide chain having the sequence of SEQ ID NO:12.

II.G.2 HSA-(G₄S)₄-GDF15

The designation “HSA-(G₄S)₄-GDF15” in the instant disclosure refers to afusion protein comprising a GDF15 polypeptide linked to an HSApolypeptide via a linker comprising the sequence of SEQ ID NO:18 thatconnects the N-terminus of the GDF15 polypeptide to the C-terminus ofthe HSA polypeptide.

In certain embodiments, a homodimer is provided comprising twoHSA-(G₄S)₄-GDF15 fusion proteins, linked via an interchain disulfidebond between their respective GDF15 regions.

More particularly, in a specific embodiment, the homodimer comprises:

(a) two HSA polypeptides (one each monomer) comprising the sequence ofSEQ ID NO:110;

(b) two GDF15 polypeptides (one each monomer) comprising the sequence ofSEQ ID NO:12, and

(c) two polypeptide linkers (one each monomer) comprising the sequenceof SEQ ID NO:18 each linking the N-terminus of a GDF15 polypeptide tothe C-terminus of an HSA polypeptide via a peptide bond.

In a preferred embodiment, the fusion protein comprises the amino acidsequence of SEQ ID NO:112, which is encoded by the nucleic acid sequenceof SEQ ID NO:111.

As discussed above, in a specific embodiment, a homodimer is providedcomprising two fusion proteins having the sequence of SEQ ID NO:112.

II.G.3 HSA-GSPAPAPGS-GDF15

The designation “HSA-(GSPAPAPGS)-GDF15” in the instant disclosure refersto a fusion protein comprising a GDF15 polypeptide linked to an HSApolypeptide via a linker comprising the sequence of SEQ ID NO:113 thatconnects the N-terminus of the GDF15 polypeptide to the C-terminus ofthe HSA polypeptide.

In certain embodiments, a homodimer is provided comprising twoHSA-(GSPAPAPGS)-GDF15 fusion proteins, linked via an interchaindisulfide bond between their respective GDF15 regions.

More particularly, in a specific embodiment, the homodimer comprises:

(a) two HSA polypeptides (one each monomer) comprising the sequence ofSEQ ID NO:110;

(b) two GDF15 polypeptides (one each monomer) comprising the sequence ofSEQ ID NO:12; and

(c) two polypeptide linkers (one each monomer) comprising the sequence:

GSPAPAPGS (SEQ ID NO:113)

each linking the N-terminus of the GDF-15 polypeptide to the C-terminusof the HSA polypeptide via a peptide bond.

In a preferred embodiment, the fusion protein comprises the amino acidsequence (linker double underlined):

(SEQ ID NO: 115) DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGSPAPAPGSARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLA KDCHCI,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 114) gatgcacacaagagtgaggttgctcatcgatttaaagatttgggagaagaaaatttcaaagccttggtgttgattgcctttgctcagtatcttcagcagtgtccatttgaagatcatgtaaaattagtgaatgaagtaactgaatttgcaaaaacatgtgttgctgatgagtcagctgaaaattgtgacaaatcacttcataccctttttggagacaaattatgcacagttgcaactcttcgtgaaacctatggtgaaatggctgactgctgtgcaaaacaagaacctgagagaaatgaatgcttcttgcaacacaaagatgacaacccaaacctcccccgattggtgagaccagaggttgatgtgatgtgcactgcttttcatgacaatgaagagacatttttgaaaaaatacttatatgaaattgccagaagacatccttacttttatgccccggaactccttttctttgctaaaaggtataaagctgcttttacagaatgttgccaagctgctgataaagctgcctgcctgttgccaaagctcgatgaacttcgggatgaagggaaggcttcgtctgccaaacagagactcaagtgtgccagtctccaaaaatttggagaaagagctttcaaagcatgggcagtagctcgcctgagccagagatttcccaaagctgagtttgcagaagtttccaagttagtgacagatcttaccaaagtccacacggaatgctgccatggagatctgcttgaatgtgctgatgacagggcggaccttgccaagtatatctgtgaaaatcaagattcgatctccagtaaactgaaggaatgctgtgaaaaacctctgttggaaaaatcccactgcattgccgaagtggaaaatgatgagatgcctgctgacttgccttcattagctgctgattttgttgaaagtaaggatgtttgcaaaaactatgctgaggcaaaggatgtcttcctgggcatgtttttgtatgaatatgcaagaaggcatcctgattactctgtcgtgctgctgctgagacttgccaagacatatgaaaccactctagagaagtgctgtgccgctgcagatcctcatgaatgctatgccaaagtgttcgatgaatttaaacctcttgtggaagagcctcagaatttaatcaaacaaaattgtgagctttttgagcagcttggagagtacaaattccagaatgcgctattagttcgttacaccaagaaagtaccccaagtgtcaactccaactcttgtagaggtctcaagaaacctaggaaaagtgggcagcaaatgttgtaaacatcctgaagcaaaaagaatgccctgtgcagaagactatctatccgtggtcctgaaccagttatgtgtgttgcatgagaaaacgccagtaagtgacagagtcaccaaatgctgcacagaatccttggtgaacaggcgaccatgcttttcagctctggaagtcgatgaaacatacgttcccaaagagtttaatgctgaaacattcaccttccatgcagatatatgcacactttctgagaaggagagacaaatcaagaaacaaactgcacttgttgagctcgtgaaacacaagcccaaggcaacaaaagagcaactgaaagctgttatggatgatttcgcagcttttgtagagaagtgctgcaaggctgacgataaggagacctgctttgccgaggagggtaaaaaacttgttgcggccagtcaggccgccttaggcttaggatccccagctccagctccaggaagcgcgcgcaacggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcatatga.

As discussed above, in a specific embodiment, a homodimer is providedcomprising two fusion proteins having the sequence of SEQ ID NO:115.

II.G.4 HSA-GS(PAPAP)₂GS-GDF15

The designation “HSA-GS(PAPAP)₂GS-GDF15” in the instant disclosurerefers to a fusion protein comprising a GDF15 polypeptide linked to anHSA polypeptide via a linker comprising the sequence of SEQ ID NO:116that connects the N-terminus of the GDF15 polypeptide to the C-terminusof the HSA polypeptide.

In certain embodiments, a homodimer is provided comprising twoHSA-GS(PAPAP)₂GS-GDF15 fusion proteins, linked via an interchaindisulfide bond between their respective GDF15 regions.

More particularly, in a specific embodiment, the homodimer comprises:

(a) two HSA polypeptides (one each monomer) comprising the sequence ofSEQ ID NO:110;

(b) two GDF15 polypeptides (one each monomer) comprising the sequence ofSEQ ID NO:12; and

(c) two polypeptide linkers (one each monomer) comprising the sequence:

GSPAPAPPAPAPGS (SEQ ID NO:116)

each linking the N-terminus of the GDF-15 polypeptide to the C-terminusof the HSA polypeptide via a peptide bond.

In a preferred embodiment, the fusion protein comprises the amino acidsequence (linker double underlined):

(SEQ ID NO: 118) DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASGAALGLGSPAPAPPAPAPGSARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTY DDLLAKDCHCI,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 117) gatgcacacaagagtgaggttgctcatcgatttaaagatttgggagaagaaaatttcaaagccttggtgttgattgcctttgctcagtatcttcagcagtgtccatttgaagatcatgtaaaattagtgaatgaagtaactgaatttgcaaaaacatgtgttgctgatgagtcagctgaaaattgtgacaaatcacttcataccctttttggagacaaattatgcacagttgcaactcttcgtgaaacctatggtgaaatggctgactgctgtgcaaaacaagaacctgagagaaatgaatgcttcttgcaacacaaagatgacaacccaaacctcccccgattggtgagaccagaggttgatgtgatgtgcactgcttttcatgacaatgaagagacatttttgaaaaaatacttatatgaaattgccagaagacatccttacttttatgccccggaactccttttctttgctaaaaggtataaagctgcttttacagaatgttgccaagctgctgataaagctgcctgcctgttgccaaagctcgatgaacttcgggatgaagggaaggcttcgtctgccaaacagagactcaagtgtgccagtctccaaaaatttggagaaagagctttcaaagcatgggcagtagctcgcctgagccagagatttcccaaagctgagtttgcagaagtttccaagttagtgacagatcttaccaaagtccacacggaatgctgccatggagatctgcttgaatgtgctgatgacagggcggaccttgccaagtatatctgtgaaaatcaagattcgatctccagtaaactgaaggaatgctgtgaaaaacctctgttggaaaaatcccactgcattgccgaagtggaaaatgatgagatgcctgctgacttgccttcattagctgctgattttgttgaaagtaaggatgtttgcaaaaactatgctgaggcaaaggatgtcttcctgggcatgtttttgtatgaatatgcaagaaggcatcctgattactctgtcgtgctgctgctgagacttgccaagacatatgaaaccactctagagaagtgctgtgccgctgcagatcctcatgaatgctatgccaaagtgttcgatgaatttaaacctcttgtggaagagcctcagaatttaatcaaacaaaattgtgagctttttgagcagcttggagagtacaaattccagaatgcgctattagttcgttacaccaagaaagtaccccaagtgtcaactccaactcttgtagaggtctcaagaaacctaggaaaagtgggcagcaaatgttgtaaacatcctgaagcaaaaagaatgccctgtgcagaagactatctatccgtggtcctgaaccagttatgtgtgttgcatgagaaaacgccagtaagtgacagagtcaccaaatgctgcacagaatccttggtgaacaggcgaccatgcttttcagctctggaagtcgatgaaacatacgttcccaaagagtttaatgctgaaacattcaccttccatgcagatatatgcacactttctgagaaggagagacaaatcaagaaacaaactgcacttgttgagctcgtgaaacacaagcccaaggcaacaaaagagcaactgaaagctgttatggatgatttcgcagcttttgtagagaagtgctgcaaggctgacgataaggagacctgctttgccgaggagggtaaaaaacttgttgcggccagtcaggccgccttaggcttaggatccccagctccagctccacccgcacctgcccctggaagcgcgcgcaacggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcatatga.

As discussed above, in a specific embodiment, a homodimer is providedcomprising two fusion proteins having the sequence of SEQ ID NO:118.

II.G.5 HSA-GSAAQAAQQGS-GDF15

The designation “HSA-GSAAQAAQQGS-GDF15 in the instant disclosure refersto a fusion protein comprising a GDF15 polypeptide linked to an HSApolypeptide via a linker comprising the sequence of SEQ ID NO:119 thatconnects the N-terminus of the GDF15 polypeptide to the C-terminus ofthe HSA polypeptide.

In certain embodiments, a homodimer is provided comprising twoHSA-GSAAQAAQQGS-GDF15 fusion proteins, linked via an interchaindisulfide bond between their respective GDF15 regions.

More particularly, in a specific embodiment, the homodimer comprises:

(a) two HSA polypeptides (one each monomer) comprising the sequence ofSEQ ID NO:110;

(b) two GDF15 polypeptides (one each monomer) comprising the sequence ofSEQ ID NO:12; and

(c) two polypeptide linkers (one each monomer) comprising the sequence:

GSAAQAAQQGS (SEQ ID NO:119)

each linking the N-terminus of the GDF15 polypeptide to the C-terminusof an HSA polypeptide via a peptide bond.

In a preferred embodiment, the fusion protein comprises the amino acidsequence (linker double underlined):

(SEQ ID NO: 121) DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGSAAQAAQQGSARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDL LAKDCHCI,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 120) gatgcacacaagagtgaggttgctcatcgatttaaagatttgggagaagaaaatttcaaagccttggtgttgattgcctttgctcagtatcttcagcagtgtccatttgaagatcatgtaaaattagtgaatgaagtaactgaatttgcaaaaacatgtgttgctgatgagtcagctgaaaattgtgacaaatcacttcataccctttttggagacaaattatgcacagttgcaactcttcgtgaaacctatggtgaaatggctgactgctgtgcaaaacaagaacctgagagaaatgaatgcttcttgcaacacaaagatgacaacccaaacctcccccgattggtgagaccagaggttgatgtgatgtgcactgcttttcatgacaatgaagagacatttttgaaaaaatacttatatgaaattgccagaagacatccttacttttatgccccggaactccttttctttgctaaaaggtataaagctgcttttacagaatgttgccaagctgctgataaagctgcctgcctgttgccaaagctcgatgaacttcgggatgaagggaaggcttcgtctgccaaacagagactcaagtgtgccagtctccaaaaatttggagaaagagctttcaaagcatgggcagtagctcgcctgagccagagatttcccaaagctgagtttgcagaagtttccaagttagtgacagatcttaccaaagtccacacggaatgctgccatggagatctgcttgaatgtgctgatgacagggcggaccttgccaagtatatctgtgaaaatcaagattcgatctccagtaaactgaaggaatgctgtgaaaaacctctgttggaaaaatcccactgcattgccgaagtggaaaatgatgagatgcctgctgacttgccttcattagctgctgattttgttgaaagtaaggatgtttgcaaaaactatgctgaggcaaaggatgtcttcctgggcatgtttttgtatgaatatgcaagaaggcatcctgattactctgtcgtgctgctgctgagacttgccaagacatatgaaaccactctagagaagtgctgtgccgctgcagatcctcatgaatgctatgccaaagtgttcgatgaatttaaacctcttgtggaagagcctcagaatttaatcaaacaaaattgtgagctttttgagcagcttggagagtacaaattccagaatgcgctattagttcgttacaccaagaaagtaccccaagtgtcaactccaactcttgtagaggtctcaagaaacctaggaaaagtgggcagcaaatgttgtaaacatcctgaagcaaaaagaatgccctgtgcagaagactatctatccgtggtcctgaaccagttatgtgtgttgcatgagaaaacgccagtaagtgacagagtcaccaaatgctgcacagaatccttggtgaacaggcgaccatgcttttcagctctggaagtcgatgaaacatacgttcccaaagagtttaatgctgaaacattcaccttccatgcagatatatgcacactttctgagaaggagagacaaatcaagaaacaaactgcacttgttgagctcgtgaaacacaagcccaaggcaacaaaagagcaactgaaagctgttatggatgatttcgcagcttttgtagagaagtgctgcaaggctgacgataaggagacctgctttgccgaggagggtaaaaaacttgttgcggccagtcaggccgccttaggcttaggatccgccgctcaggctgcacagcaaggaagcgcgcgcaacggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcatatga.

As discussed above, in a specific embodiment, a homodimer is providedcomprising two fusion proteins having the sequence of SEQ ID NO:121.

II.G.6 HSA-GS(AAQAAQQ)₂GS-GDF15

The designation “HSA-GS(AAQAAQQ)₂GS-GDF15” in the instant disclosurerefers to a fusion protein comprising a GDF15 polypeptide linked to anHSA polypeptide via a linker comprising the sequence of SEQ ID NO:122that connects the N-terminus of the GDF15 polypeptide to the C-terminusof the HSA polypeptide.

In certain embodiments, a homodimer is provided comprising twoHSA-GS(AAQAAQQ)₂GS-GDF15 fusion proteins, linked via an interchaindisulfide bond between their respective GDF15 regions.

More particularly, in a specific embodiment, the homodimer comprises:

(a) two HSA polypeptides (one each monomer) comprising the sequence ofSEQ ID NO:110;

(b) two GDF15 polypeptides (one each monomer) comprising the sequence ofSEQ ID NO:12; and

(c) two polypeptide linkers (one each monomer) comprising the sequence:

GSAAQAAQQAAQAAQQGS (SEQ ID NO:122)

each linking the N-terminus of the GDF-15 polypeptide to the C-terminusof the HSA polypeptide via a peptide bond.

In a preferred embodiment, the fusion protein comprises the amino acidsequence (linker double underlined):

(SEQ ID NO: 124) DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGSAAQAAQQAAQAAQQGSARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVS LQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 123) gatgcacacaagagtgaggttgctcatcgatttaaagatttgggagaagaaaatttcaaagccttggtgttgattgcctttgctcagtatcttcagcagtgtccatttgaagatcatgtaaaattagtgaatgaagtaactgaatttgcaaaaacatgtgttgctgatgagtcagctgaaaattgtgacaaatcacttcataccctttttggagacaaattatgcacagttgcaactcttcgtgaaacctatggtgaaatggctgactgctgtgcaaaacaagaacctgagagaaatgaatgcttcttgcaacacaaagatgacaacccaaacctcccccgattggtgagaccagaggttgatgtgatgtgcactgcttttcatgacaatgaagagacatttttgaaaaaatacttatatgaaattgccagaagacatccttacttttatgccccggaactccttttctttgctaaaaggtataaagctgcttttacagaatgttgccaagctgctgataaagctgcctgcctgttgccaaagctcgatgaacttcgggatgaagggaaggcttcgtctgccaaacagagactcaagtgtgccagtctccaaaaatttggagaaagagctttcaaagcatgggcagtagctcgcctgagccagagatttcccaaagctgagtttgcagaagtttccaagttagtgacagatcttaccaaagtccacacggaatgctgccatggagatctgcttgaatgtgctgatgacagggcggaccttgccaagtatatctgtgaaaatcaagattcgatctccagtaaactgaaggaatgctgtgaaaaacctctgttggaaaaatcccactgcattgccgaagtggaaaatgatgagatgcctgctgacttgccttcattagctgctgattttgttgaaagtaaggatgtttgcaaaaactatgctgaggcaaaggatgtcttcctgggcatgtttttgtatgaatatgcaagaaggcatcctgattactctgtcgtgctgctgctgagacttgccaagacatatgaaaccactctagagaagtgctgtgccgctgcagatcctcatgaatgctatgccaaagtgttcgatgaatttaaacctcttgtggaagagcctcagaatttaatcaaacaaaattgtgagctttttgagcagcttggagagtacaaattccagaatgcgctattagttcgttacaccaagaaagtaccccaagtgtcaactccaactcttgtagaggtctcaagaaacctaggaaaagtgggcagcaaatgttgtaaacatcctgaagcaaaaagaatgccctgtgcagaagactatctatccgtggtcctgaaccagttatgtgtgttgcatgagaaaacgccagtaagtgacagagtcaccaaatgctgcacagaatccttggtgaacaggcgaccatgcttttcagctctggaagtcgatgaaacatacgttcccaaagagtttaatgctgaaacattcaccttccatgcagatatatgcacactttctgagaaggagagacaaatcaagaaacaaactgcacttgttgagctcgtgaaacacaagcccaaggcaacaaaagagcaactgaaagctgttatggatgatttcgcagcttttgtagagaagtgctgcaaggctgacgataaggagacctgctttgccgaggagggtaaaaaacttgttgcggccagtcaggccgccttaggcttaggatccgccgctcaggctgcacagcaagcagcccaagcagctcagcagggaagcgcgcgcaacggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcatatga.

As discussed above, in a specific embodiment, a homodimer is providedcomprising two fusion proteins having the sequence of SEQ ID NO:124.

II.G.7 HSA-GGNAEAAAKEAAAKEAAAKAGG-GDF15

The designation “HSA-GGNAEAAAKEAAAKEAAAKAGG-GDF15” in the instantdisclosure refers to a fusion protein comprising a GDF15 polypeptidelinked to an HSA polypeptide via a linker comprising the sequence of SEQID NO:125 that connects the N-terminus of the GDF15 polypeptide to theC-terminus of the HSA polypeptide.

In certain embodiments, a homodimer is provided comprising twoHSA-GGNAEAAAKEAAAKEAAAKAGG-GDF 15 fusion proteins, linked via aninterchain disulfide bond between their respective GDF15 regions.

More particularly, in a specific embodiment, the homodimer comprises:

(a) two HSA polypeptides (one each monomer) comprising the sequence ofSEQ ID NO:110;

(b) two GDF15 polypeptides (one each monomer) comprising the sequence ofSEQ ID NO:12; and

(c) two polypeptide linkers (one each monomer) comprising the sequence:

GGNAEAAAKEAAAKEAAAKAGG (SEQ ID NO:125)

each linking the N-terminus of the GDF-15 polypeptide to the C-terminusof the HSA polypeptide via a peptide bond.

In a preferred embodiment, the fusion protein comprises the amino acidsequence (linker double underlined):

(SEQ ID NO: 127) DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGGNAEAAAKEAAAKEAAAKEAAAKAGGARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 126) gatgcacacaagagtgaggttgctcatcgatttaaagatttgggagaagaaaatttcaaagccttggtgttgattgcctttgctcagtatcttcagcagtgtccatttgaagatcatgtaaaattagtgaatgaagtaactgaatttgcaaaaacatgtgttgctgatgagtcagctgaaaattgtgacaaatcacttcataccctttttggagacaaattatgcacagttgcaactcttcgtgaaacctatggtgaaatggctgactgctgtgcaaaacaagaacctgagagaaatgaatgcttcttgcaacacaaagatgacaacccaaacctcccccgattggtgagaccagaggttgatgtgatgtgcactgcttttcatgacaatgaagagacatttttgaaaaaatacttatatgaaattgccagaagacatccttacttttatgccccggaactccttttctttgctaaaaggtataaagctgcttttacagaatgttgccaagctgctgataaagctgcctgcctgttgccaaagctcgatgaacttcgggatgaagggaaggcttcgtctgccaaacagagactcaagtgtgccagtctccaaaaatttggagaaagagctttcaaagcatgggcagtagctcgcctgagccagagatttcccaaagctgagtttgcagaagtttccaagttagtgacagatcttaccaaagtccacacggaatgctgccatggagatctgcttgaatgtgctgatgacagggcggaccttgccaagtatatctgtgaaaatcaagattcgatctccagtaaactgaaggaatgctgtgaaaaacctctgttggaaaaatcccactgcattgccgaagtggaaaatgatgagatgcctgctgacttgccttcattagctgctgattttgttgaaagtaaggatgtttgcaaaaactatgctgaggcaaaggatgtcttcctgggcatgtttttgtatgaatatgcaagaaggcatcctgattactctgtcgtgctgctgctgagacttgccaagacatatgaaaccactctagagaagtgctgtgccgctgcagatcctcatgaatgctatgccaaagtgttcgatgaatttaaacctcttgtggaagagcctcagaatttaatcaaacaaaattgtgagctttttgagcagcttggagagtacaaattccagaatgcgctattagttcgttacaccaagaaagtaccccaagtgtcaactccaactcttgtagaggtctcaagaaacctaggaaaagtgggcagcaaatgttgtaaacatcctgaagcaaaaagaatgccctgtgcagaagactatctatccgtggtcctgaaccagttatgtgtgttgcatgagaaaacgccagtaagtgacagagtcaccaaatgctgcacagaatccttggtgaacaggcgaccatgcttttcagctctggaagtcgatgaaacatacgttcccaaagagtttaatgctgaaacattcaccttccatgcagatatatgcacactttctgagaaggagagacaaatcaagaaacaaactgcacttgttgagctcgtgaaacacaagcccaaggcaacaaaagagcaactgaaagctgttatggatgatttcgcagcttttgtagagaagtgctgcaaggctgacgataaggagacctgctttgccgaggagggtaaaaaacttgttgcggccagtcaggccgccttaggcttaggaggcaacgccgaggctgccgctaaggaagccgctgccaaggaggccgcagcaaaagaggctgcagctaaggccggaggagcgcgcaacggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcatatga.

As discussed above, in a specific embodiment, a homodimer is providedcomprising two fusion proteins having the sequence of SEQ ID NO:127.

II.G.8 HSA-(G₄S)₆-GDF15

The designation “HSA-(G₄S)₆-GDF15” in the instant disclosure refers to afusion protein comprising a GDF15 polypeptide linked to an HSApolypeptide via a linker comprising the sequence of SEQ ID NO:128 thatconnects the N-terminus of the GDF15 polypeptide to the C-terminus ofthe HSA polypeptide.

In certain embodiments, a homodimer is provided comprising twoHSA-(G₄S)₆-GDF15 fusion proteins, linked via an interchain disulfidebond between their respective GDF15 regions.

More particularly, in a specific embodiment, the homodimer comprises:

(a) two HSA polypeptides (one each monomer) comprising the sequence ofSEQ ID NO:110;

(b) two GDF15 polypeptides (one each monomer) comprising the sequence ofSEQ ID NO:12; and

(c) two polypeptide linkers (one each monomer) comprising the sequence:

GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO:128)

each linking the N-terminus of the GDF-15 polypeptide to the C-terminusof the HSA polypeptide via a peptide bond.

In a preferred embodiment, the fusion protein comprises the amino acidsequence (linker double underlined):

(SEQ ID NO: 130) DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFADAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 129) gatgcacacaagagtgaggttgctcatcgatttaaagatttgggagaagaaaatttcaaagccttggtgttgattgcctttgctcagtatcttcagcagtgtccatttgaagatcatgtaaaattagtgaatgaagtaactgaatttgcaaaaacatgtgttgctgatgagtcagctgaaaattgtgacaaatcacttcataccctttttggagacaaattatgcacagttgcaactcttcgtgaaacctatggtgaaatggctgactgctgtgcaaaacaagaacctgagagaaatgaatgcttcttgcaacacaaagatgacaacccaaacctcccccgattggtgagaccagaggttgatgtgatgtgcactgcttttcatgacaatgaagagacatttttgaaaaaatacttatatgaaattgccagaagacatccttacttttatgccccggaactccttttctttgctaaaaggtataaagctgcttttacagaatgttgccaagctgctgataaagctgcctgcctgttgccaaagctcgatgaacttcgggatgaagggaaggcttcgtctgccaaacagagactcaagtgtgccagtctccaaaaatttggagaaagagctttcaaagcatgggcagtagctcgcctgagccagagatttcccaaagctgagtttgcagaagtttccaagttagtgacagatcttaccaaagtccacacggaatgctgccatggagatctgcttgaatgtgctgatgacagggcggaccttgccaagtatatctgtgaaaatcaagattcgatctccagtaaactgaaggaatgctgtgaaaaacctctgttggaaaaatcccactgcattgccgaagtggaaaatgatgagatgcctgctgacttgccttcattagctgctgattttgttgaaagtaaggatgtttgcaaaaactatgctgaggcaaaggatgtcttcctgggcatgtttttgtatgaatatgcaagaaggcatcctgattactctgtcgtgctgctgctgagacttgccaagacatatgaaaccactctagagaagtgctgtgccgctgcagatcctcatgaatgctatgccaaagtgttcgatgaatttaaacctcttgtggaagagcctcagaatttaatcaaacaaaattgtgagctttttgagcagcttggagagtacaaattccagaatgcgctattagttcgttacaccaagaaagtaccccaagtgtcaactccaactcttgtagaggtctcaagaaacctaggaaaagtgggcagcaaatgttgtaaacatcctgaagcaaaaagaatgccctgtgcagaagactatctatccgtggtcctgaaccagttatgtgtgttgcatgagaaaacgccagtaagtgacagagtcaccaaatgctgcacagaatccttggtgaacaggcgaccatgcttttcagctctggaagtcgatgaaacatacgttcccaaagagtttaatgctgaaacattcaccttccatgcagatatatgcacactttctgagaaggagagacaaatcaagaaacaaactgcacttgttgagctcgtgaaacacaagcccaaggcaacaaaagagcaactgaaagctgttatggatgatttcgcagcttttgtagagaagtgctgcaaggctgacgataaggagacctgctttgccgaggagggtaaaaaacttgttgcggccagtcaggccgccttaggcttaggaggtggtggctctggaggcggtggaagcggaggcggtggatccggaggcggtggaagcggaggtggtggatctggaggcggtggaagcgcgcgcaacggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcatatga.

As discussed above, in a specific embodiment, a homodimer is providedcomprising two fusion proteins having the sequence of SEQ ID NO:130.

II.G.9 HSA-GS(AAQAAQQ)₂GS-GDF15(N3D)

The designation “HSA-GS(AAQAAQQ)₂GS-GDF15(N3D)” in the instantdisclosure refers to a fusion protein comprising a GDF15(N3D)polypeptide linked to an HSA polypeptide via a linker comprising thesequence of SEQ ID NO:122 that connects the N-terminus of the GDF15(N3D)polypeptide to the C-terminus of the HSA polypeptide.

In certain embodiments, a homodimer is provided comprising twoHSA-GS(AAQAAQQ)₂GS-GDF15(N3D) fusion proteins, linked via an interchaindisulfide bond between their respective GDF15 regions.

More particularly, in a specific embodiment, the homodimer comprises:

(a) two HSA polypeptides (one each monomer) comprising the sequence ofSEQ ID NO:110;

(b) two GDF15(N3D) polypeptides (one each monomer) comprising thesequence of SEQ ID NO:52; and

(c) two polypeptide linkers (one each monomer) comprising the sequenceof SEQ ID NO:122 each linking the N-terminus of a GDF15(N3D) polypeptideto the C-terminus of an HSA polypeptide via a peptide bond.

In a preferred embodiment, the fusion protein comprises the amino acidsequence (linker double underlined):

(SEQ ID NO: 242) DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGSAAQAAQQAAQAAQQGSARDGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVS LQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 241) gatgcacacaagagtgaggttgctcatcgatttaaagatttgggagaagaaaatttcaaagccttggtgttgattgcctttgctcagtatcttcagcagtgtccatttgaagatcatgtaaaattagtgaatgaagtaactgaatttgcaaaaacatgtgttgctgatgagtcagctgaaaattgtgacaaatcacttcataccctttttggagacaaattatgcacagttgcaactcttcgtgaaacctatggtgaaatggctgactgctgtgcaaaacaagaacctgagagaaatgaatgcttcttgcaacacaaagatgacaacccaaacctcccccgattggtgagaccagaggttgatgtgatgtgcactgcttttcatgacaatgaagagacatttttgaaaaaatacttatatgaaattgccagaagacatccttacttttatgccccggaactccttttctttgctaaaaggtataaagctgcttttacagaatgttgccaagctgctgataaagctgcctgcctgttgccaaagctcgatgaacttcgggatgaagggaaggcttcgtctgccaaacagagactcaagtgtgccagtctccaaaaatttggagaaagagctttcaaagcatgggcagtagctcgcctgagccagagatttcccaaagctgagtttgcagaagtttccaagttagtgacagatcttaccaaagtccacacggaatgctgccatggagatctgcttgaatgtgctgatgacagggcggaccttgccaagtatatctgtgaaaatcaagattcgatctccagtaaactgaaggaatgctgtgaaaaacctctgttggaaaaatcccactgcattgccgaagtggaaaatgatgagatgcctgctgacttgccttcattagctgctgattttgttgaaagtaaggatgtttgcaaaaactatgctgaggcaaaggatgtcttcctgggcatgtttttgtatgaatatgcaagaaggcatcctgattactctgtcgtgctgctgctgagacttgccaagacatatgaaaccactctagagaagtgctgtgccgctgcagatcctcatgaatgctatgccaaagtgttcgatgaatttaaacctcttgtggaagagcctcagaatttaatcaaacaaaattgtgagctttttgagcagcttggagagtacaaattccagaatgcgctattagttcgttacaccaagaaagtaccccaagtgtcaactccaactcttgtagaggtctcaagaaacctaggaaaagtgggcagcaaatgttgtaaacatcctgaagcaaaaagaatgccctgtgcagaagactatctatccgtggtcctgaaccagttatgtgtgttgcatgagaaaacgccagtaagtgacagagtcaccaaatgctgcacagaatccttggtgaacaggcgaccatgcttttcagctctggaagtcgatgaaacatacgttcccaaagagtttaatgctgaaacattcaccttccatgcagatatatgcacactttctgagaaggagagacaaatcaagaaacaaactgcacttgttgagctcgtgaaacacaagcccaaggcaacaaaagagcaactgaaagctgttatggatgatttcgcagcttttgtagagaagtgctgcaaggctgacgataaggagacctgctttgccgaggagggtaaaaaacttgttgcggccagtcaggccgccttaggcttaggatccgccgctcaggctgcacagcaagcagcccaagcagctcagcagggaagcgcgcgcgacggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgca ta.

As discussed above, in a specific embodiment, a homodimer is providedcomprising two fusion proteins having the sequence of SEQ ID NO:242.

II.H. Constructs with Mutations to Address Affinity for FcγR Binding

It was found that certain charged pair (delHinge) multimers exhibitedFcγ receptor (FcγR) binding, particularly to FcγRI and FcγRIII. See,e.g., Example 7. In some cases, the FcγR binding affinity was comparableto that of observed in multimers comprising a hinge region. This wasunexpected, because the Fcγ receptor interacts with the hinge region andthese multimers were delHinge, as described above, lacking all or partof the hinge region. Mutational analyses of the Fc residues involved inbinding to FcγR suggest that the main interaction site is localized inthe hinge region and CH2 domain (Tamm A, 1997, Int. Rev. Immunol.16:57-85). See also, Radaev S, et al., J. Biol. Chem. 276:16469-16477;Sondermann, P, et al., 2000, Nature 406:267-273.

Antibody-dependent cellular cytotoxicity (ADCC), an immune responsemediated primarily by natural killer (NK) cells in humans, depends onthe interaction of FcγRs, especially FcγRIIIA in humans, with the Fcdomain of an antibody or Fc-containing protein. In ADCC, Fc binding toFcγRIII on the surface of an NK cell activates the NK cell, whichreleases perforins and granzymes.

Accordingly, constructs are provided having additional modifications tomodulate the interaction of the construct with the FcγR. In one seriesof embodiments, an asparagine to glycine mutation (N297G) is introducedto a native Fc or Fc variant, including the various Fc domains describedabove. The N297G mutation removes a conserved N-glycosylation site at inthe CH2 domain.

In another series of embodiments, additional N-terminal amino acidresidues are deleted from an Fc domain from which all or a portion ofthe hinge region has been removed. For example, the amino acid sequence:

(SEQ ID NO: 303) GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGKis provided by deletion of amino acid residues N-terminal to G236 of theFc domain of wild-type IgG1. In some embodiments, the C-terminal lysine(K447) optionally may be deleted from this Fc variant. The amino acidsequence:

(SEQ ID NO: 304) GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGKis provided by deletion of amino acid residues N-terminal to G237 fromthe Fc domain of wild-type IgG1. In some embodiments, the C-terminallysine (K447) optionally may be deleted from this Fc variant. The aminoacid sequence:

(SEQ ID NO: 305) PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGKis provided by deletion of amino acid residues N-terminal to P238 of theFc domain of wild-type IgG1. In some embodiments, the C-terminal lysine(K447) optionally may be deleted from this Fc variant.

In one embodiment, a DhCpmFc(−) domain into which an asparagine toglycine mutation (N297G) has been introduced is provided (referred toherein as a “DhCpmFc(−)(N297G)” domain). In another embodiment, aDhCpmFc(+) domain into which an asparagine to glycine mutation (N297G)has been introduced is provided (referred to herein as a“DhCpmFc(+)(N297G)” domain).

In another embodiment, a DhCpmFc(−)(Y349C) domain into which anasparagine to glycine mutation (N297G) has been introduced is provided(referred to herein as a “DhCpmFc(−)(N297G)(Y349C)” domain). In anotherembodiment, a DhCpmFc(+)(S354C) domain into which an asparagine toglycine mutation (N297G) has been introduced is provided (referred toherein as a “DhCpmFc(+)(N297G)(S354C)” domain).

In another embodiment, a DhCpmFc(+)(Y349C) domain into which anasparagine to glycine mutation (N297G) has been introduced is provided(referred to herein as a “DhCpmFc(+)(N297G)(Y349C)” domain). In anotherembodiment, a DhCpmFc(−)(S354C) domain into which an asparagine toglycine mutation (N297G) has been introduced is provided (referred toherein as “DhCpmFc(−)(N297G)(S354C)” domain).

In another embodiment, a DhCpmFc(+)(L351C) domain into which anasparagine to glycine mutation (N297G) has been introduced is provided(referred to herein as a “DhCpmFc(+)(N297G)(L351C)” domain). In anotherembodiment, a DhCpmFc(−)(L351C) domain into which an asparagine toglycine mutation (N297G) has been introduced is provided (referred toherein as a “DhCpmFc(−)(N297G)(L351C)” domain).

In another embodiment, a DhCpmFc(−) domain into which an alanine tocysteine mutation (A287C) has been introduced is provided (referred toherein as a “DhCpmFc(−)(A287C)” domain). In another embodiment, aDhCpmFc(+) domain into which an alanine to cysteine mutation (A287C) hasbeen introduced is provided (referred to herein as a “DhCpmFc(+)(A287C)”domain).

In another embodiment, a DhCpmFc(−) domain into which a leucine tocysteine mutation (L306C) has been introduced is provided (referred toherein as a “DhCpmFc(−)(L306C)” domain). In another embodiment, aDhCpmFc(+) domain into which a leucine to cysteine mutation (L306C) hasbeen introduced is provided (referred to herein as a “DhCpmFc(+)(L306C)”domain).

In another embodiment, a DhCpmFc(−)(A287C) domain into which a tyrosineto cysteine mutation (Y349C) has been introduced is provided (referredto herein as a “DhCpmFc(−)(A287C)(Y349C)” domain). In anotherembodiment, a DhCpmFc(+)(A287C) domain into which a tyrosine to cysteinemutation (Y349C) has been introduced is provided (referred to herein asa “DhCpmFc(+)(A287C)(Y349C)” domain).

In another embodiment, a DhCpmFc(−)(A287C) domain into which a serine tocysteine mutation (S354C) has been introduced is provided (referred toherein as a “DhCpmFc(−)(A287C)(S354C)” domain). In another embodiment, aDhCpmFc(+)(A287C) domain into which a serine to cysteine mutation(S354C) has been introduced is provided (referred to herein as a“DhCpmFc(+)(A287C)(S354C)” domain).

In another embodiment, a DhCpmFc(−)(L306C) domain into which a tyrosineto cysteine mutation (Y349C) has been introduced is provided (referredto herein as a “DhCpmFc(−)(L306C)(Y349C)” domain). In anotherembodiment, a DhCpmFc(+)(L306C) domain into which a tyrosine to cysteinemutation (Y349C) has been introduced is provided (referred to herein asa “DhCpmFc(+)(L306C)(Y349C)” domain).

In another embodiment, a DhCpmFc(−)(L306C) domain into which a serine tocysteine mutation (S354C) has been introduced is provided (referred toherein as a “DhCpmFc(−)(L306C)(S354C)” domain). In another embodiment, aDhCpmFc(+)(L306C) domain into which a serine to cysteine mutation(S354C) has been introduced is provided (referred to herein as a“DhCpmFc(+)(A287C)(L306C)” domain).

In another embodiment, a DhCpmFc(−) domain from which the N-terminal 7amino acids have been removed is provided (referred to herein as a“Dh2CpmFc(−)” domain). In another embodiment, a DhCpmFc(+) domain fromwhich the N-terminal 7 amino acids have been removed is provided(referred to herein as a “Dh2CpmFc(+)” domain).

In another embodiment, a DhCpmFc(−)(Y349C) domain from which theN-terminal 7 amino acids have been removed is provided (referred toherein as a “Dh2CpmFc(−)(Y349C)” domain). In another embodiment, aDhCpmFc(+)(S354C) domain from which the N-terminal 7 amino acids havebeen removed is provided (referred to herein as a “Dh2CpmFc(+)(S354C)”domain).

In another embodiment, a DhCpmFc(+)(Y349C) domain from which theN-terminal 7 amino acids have been removed is provided (referred toherein as a “Dh2CpmFc(+)(Y349C)” domain). In another embodiment, aDhCpmFc(−)(S354C) domain from which the N-terminal 7 amino acids havebeen removed is provided (referred to herein as a “Dh2CpmFc(−) (S354C)”domain).

In another embodiment, a CpmFc(+) domain into which an asparagine toglycine mutation (N297G) has been introduced is provided (referred toherein as a “CpmFc(+)(N297G)” domain). In another embodiment, a CpmFc(−)domain into which an asparagine to glycine mutation (N297G) has beenintroduced is provided (referred to herein as a “CpmFc(−)(N297G)”domain).

In another embodiment, a Dh2CpmFc(+) domain into which an asparagine toglycine mutation (N297G) has been introduced is provided (referred toherein as a “Dh2CpmFc(+)(N297G)” domain). In another embodiment, aDh2CpmFc(−) domain into which an asparagine to glycine mutation (N297G)has been introduced is provided (referred to herein as a“Dh2CpmFc(−)(N297G)” domain).

In another embodiment, a Dh2CpmFc(−)(N297G) domain into which an alanineto cysteine mutation (A287C) has been introduced is provided (referredto herein as a “Dh2CpmFc(−)(N297G)(A287C)” domain). In anotherembodiment, Dh2CpmFc(+)(N297G) domain into which a leucine to cysteinemutation (L306C) has been introduced is provided (referred to herein asa “Dh2CpmFc(+)(N297G)(L306C)” domain).

In another embodiment, a Dh2CpmFc(−)(N297G)(A287C) domain into which atyrosine to cysteine mutation (Y349C) has been introduced is provided(referred to herein as a “Dh2CpmFc(−)(N297G)(A287C)(Y349C)” domain). Inanother embodiment, a Dh2CpmFc(+)(N297G)(L306C) domain into which aserine to cysteine mutation (S354C) has been introduced is provided(referred to herein as a “Dh2CpmFc(+)(N297G)(L306C)(S354C)” domain).

In another embodiment, a Dh2CpmFc(−) domain onto which two glycines havebeen added to the N-terminus is provided (referred to herein as a“GG-Dh2CpmFc(−)(N297G)” domain). In another embodiment, a Dh2CpmFc(+)domain onto which two glycines have been added to the N-terminus isprovided (referred to herein as a “GG-Dh2CpmFc(+)(N297G)” domain).

In another embodiment, a GG-Dh2CpmFc(−) domain into which a tyrosine tocysteine mutation (Y349C) have been introduced is provided (referred toherein as a “GG-Dh2CpmFc(−)(Y349C)” domain). In another embodiment, aGG-Dh2CpmFc(+) domain into which a serine to cysteine mutation (S354C)has been introduced is provided (referred to herein as a“GG-Dh2CpmFc(+)(S354C)” domain).

In another embodiment, a GG-Dh2CpmFc(+) domain into which a tyrosine tocysteine mutation (Y349C) has been introduced is provided (referred toherein as a “GG-Dh2CpmFc(+)(Y349C)” domain). In another embodiment, aGG-Dh2CpmFc(−) domain into which a serine to cysteine mutation (S354C)has been introduced is provided (referred to herein as a“GG-Dh2CpmFc(−)(S354C)” domain).

In another embodiment, a Dh2CpmFc(−) domain onto which a glycine hasbeen added to the N-terminus is provided (referred to herein as a“Dh3CpmFc(−)(N297G)” domain). In another embodiment, a Dh2CpmFc(+)domain onto which a glycine has been added to the N-terminus is provided(referred to herein as a “Dh3CpmFc(+)” domain).

In another embodiment, a Dh3CpmFc(−) domain into which a tyrosine tocysteine mutation (Y349C) has been introduced is provided (referred toherein as a “Dh3CpmFc(−)(Y349C)” domain). In another embodiment, aDh3CpmFc(+) domain into which a serine to cysteine mutation (S354C) hasbeen introduced is provided (referred to herein as a“Dh3CpmFc(+)(S354C)” domain).

In another embodiment, an asparagine to glycine mutation (N297G) isintroduced into a DhMonoFc domain (referred to herein as“DhMonoFc(N297G)”).

II.H.1 DhCpmFc(−)(N297G)-GDF15(Ndel3):DhCpmFc(+)(N297G)

The designation “DhCpmFc(−)(N297G)-GDF15(Ndel3):DhCpmFc(+)(N297G)” inthe instant disclosure refers to a heterodimer comprising (i) a firstpolypeptide chain comprising a GDF15(Ndel3) polypeptide, the N-terminusof which is linked directly to the C-terminus of a DhCpmFc(−)(N297G)domain, and (ii) a second polypeptide chain comprising aDhCpmFc(+)(N297G) domain.

In certain embodiments, a tetramer is provided, comprising a dimer oftwo DhCpmFc(−)(N297G)-GDF15(Ndel3):DhCpmFc(+)(N297G) heterodimers inwhich the two first polypeptide chains of each heterodimer are linkedvia an interchain disulfide bond between their respective GDF15 regions.

More particularly, in a specific embodiment, the tetramer comprises:

(a) two DhCpmFc(+)(N297G) domains (one each heterodimer) comprising thesequence:

(SEQ ID NO: 287) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPG,

(b) two DhCpmFc(−)(N297G) domains (one each heterodimer) comprising thesequence:

(SEQ ID NO: 132) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPG,and

(c) two GDF15(Ndel3) polypeptides (one each heterodimer) comprising thesequence of SEQ ID NO:55.

In a preferred embodiment, the first polypeptide chain comprises theamino acid sequence:

(SEQ ID NO: 134) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 133) gcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacgggagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcata.

In a preferred embodiment, the second polypeptide chain comprises theamino acid sequence:

(SEQ ID NO: 131) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 135) gcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacgggagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccggaaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctgaagtccgacggctccttcttcctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaa atga.

As discussed above, a tetramer is provided comprising two polypeptidechains comprising the sequence of SEQ ID NO:134 and two polypeptidechains comprising the sequence of SEQ ID NO:131.

II.H.2 DhCpmFc(−)(N297G)-GDF15(N3D):DhCpmFc(+)(N297G)

The designation “DhCpmFc(−)(N297G)-GDF15(N3D):DhCpmFc(+)(N297G)” in theinstant disclosure refers to a heterodimer comprising (i) a firstpolypeptide chain comprising a GDF15(N3D) sequence, the N-terminus ofwhich is linked directly to the C-terminus of a DhCpmFc(−)(N297G)domain, and (ii) a second polypeptide chain comprising aDhCpmFc(+)(N297G) domain.

In certain embodiments, a tetramer is provided, comprising a dimer oftwo DhCpmFc(−)(N297G)-GDF15(N3D):DhCpmFc(+)(N297G) heterodimers in whichthe two first polypeptide chains of each heterodimer are linked via aninterchain disulfide bond between their respective GDF15 regions.

More particularly, in a specific embodiment, the tetramer comprises:

(a) two DhCpmFc(+)(N297G) domains (one each heterodimer) comprising thesequence of SEQ ID NO:287,

(b) two DhCpmFc(−)(N297G) domains (one each heterodimer) comprising thesequence of SEQ ID NO:132, and

(c) two GDF15(N3D) polypeptides (one each heterodimer) comprising thesequence of SEQ ID NO:52.

In a preferred embodiment, the first polypeptide chain comprises theamino acid sequence:

(SEQ ID NO: 137) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGARDGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 136) gcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacgggagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtgcgcgcgacggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcata.

In a preferred embodiment, the second polypeptide chain comprises theamino acid sequence of SEQ ID NO:131, which is encoded by the nucleicacid sequence of SEQ ID NO:135.

As discussed above, a tetramer is provided comprising two polypeptidechains comprising the sequence of SEQ ID NO:137 and two polypeptidechains comprising the sequence of SEQ ID NO:131.

II.H.3 DhCpmFc(−)(N297G)-G₄-GDF15(N3D):DhCpmFc(+)(N297G)

The designation “DhCpmFc(−)(N297G)-G₄-GDF15(N3D):DhCpmFc(+)(N297G)” inthe instant disclosure refers to a heterodimer comprising (i) a firstpolypeptide chain comprising a GDF15(N3D) polypeptide linked to aDhCpmFc(−)(N297G) domain via a linker comprising the sequence of SEQ IDNO:58 that connects the N-terminus of the GDF15(N3D) polypeptide to theC-terminus of the DhCpmFc(−)(N297G) domain and (ii) a second polypeptidechain comprising a DhCpmFc(+)(N297G) domain.

In certain embodiments, a tetramer is provided, comprising a dimer oftwo DhCpmFc(−)(N297G)-G₄-GDF15(N3D):DhCpmFc(+)(N297G) heterodimers inwhich the two first polypeptide chains of each heterodimer are linkedvia an interchain disulfide bond between their respective GDF15 regions.

More particularly, in a specific embodiment, the tetramer comprises:

(a) two DhCpmFc(+)(N297G) domains (one each heterodimer) comprising thesequence of SEQ ID NO:287,

(b) two DhCpmFc(−)(N297G) domains (one each heterodimer) comprising thesequence of SEQ ID NO:132,

(c) two GDF15(N3D) polypeptides (one each heterodimer) comprising thesequence of SEQ ID NO:52, and

(d) two polypeptide linkers (one each heterodimer) comprising thesequence of SEQ ID NO:58 each linking the N-terminus of a GDF15(N3D)polypeptide to the C-terminus of a DhCpmFc(−)(N297G) domain via apeptide bond.

In a preferred embodiment, the first polypeptide chain comprises theamino acid sequence:

(SEQ ID NO: 139) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGARDGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 138) gcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacgggagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtggaggtggtggagcgcgcgacggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcata.

In a preferred embodiment, the second polypeptide chain comprises theamino acid sequence of SEQ ID NO:131, which is encoded by the nucleicacid sequence of SEQ ID NO:135.

As discussed above, a tetramer is provided comprising two polypeptidechains comprising the sequence of SEQ ID NO:139 and two polypeptidechains comprising the sequence of SEQ ID NO:131.

II.H.4 DhCpmFc(−)(N297G)(Y349C)-GDF15(Ndel3):DhCpmFc(+)(N297G)(S354C)

The designation“DhCpmFc(−)(N297G)(Y349C)-GDF15(Ndel3):DhCpmFc(+)(N297G)(S354C)” in theinstant disclosure refers to a heterodimer comprising (i) a firstpolypeptide chain comprising a GDF15(Ndel3) polypeptide, the N-terminusof which is linked directly to the C-terminus of aDhCpmFc(−)(N297G)(Y349C) domain, and (ii) a second polypeptide chaincomprising a DhCpmFc(+)(N297G)(S345C) domain. The cysteine clampmutations allow the first and second polypeptide chains to be linked viaan interchain disulfide bond between C349 of the first polypeptide chainand C354 of the second polypeptide chain.

In certain embodiments, a tetramer is provided, comprising a dimer oftwo DhCpmFc(−)(N297G)(Y349C)-GDF15(Ndel3):DhCpmFc(+)(N297G)(S354C)heterodimers in which the two first polypeptide chains of eachheterodimer are linked via an interchain disulfide bond between theirrespective GDF15 regions.

More particularly, in a specific embodiment, the tetramer comprises:

(a) two DhCpmFc(+)(N297G)(S354C) domains (one each heterodimer)comprising the sequence:

(SEQ ID NO: 288) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPG,

(b) two DhCpmFc(−)(N297G)(Y349C) domains (one each heterodimer)comprising the sequence:

(SEQ ID NO: 141) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPG,and

(c) two GDF15(Ndel3) polypeptides (one each heterodimer) comprising thesequence of SEQ ID NO:55.

In a preferred embodiment, the first polypeptide chain comprises theamino acid sequence:

(SEQ ID NO: 143) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 142) gcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacggcagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtgcaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcata.

In a preferred embodiment, the second polypeptide chain comprises theamino acid sequence:

(SEQ ID NO: 140) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 144) gccccagagctgcttggtggaccatccgtgttcctgtttcctccaaagccgaaggacaccctgatgatctcaagaactccggaagtgacttgcgtcgtcgtggacgtgtcacatgaggatccagaggtcaagttcaattggtatgtggacggagtggaagtgcataacgccaagaccaaaccccgcgaagaacagtacgggagcacctaccgcgtggtgagcgtccttactgtgctccaccaggactggcttaatgggaaggaatacaagtgtaaggtgtccaacaaggccctccccgctcccatcgaaaagaccatctcaaaggcaaaggggcaaccaagggaacctcaagtgtacaccctgcctccgtgcaggaaggagatgaccaagaaccaggtcagcctgacttgtctcgtgaagggcttctatcccagcgatattgctgtggaatgggagtcaaatggccagcccgagaataactacaaaactaccccacccgtgctgaaatctgatgggtccttcttcctttactccaagctgaccgtggacaagagccgctggcaacaaggcaatgtctttagctgctcagtgatgcatgaggctctccataatcactacactcagaagtcactgtccctgtctccgggtaa a.

As discussed above, a tetramer is provided comprising two polypeptidechains comprising the sequence of SEQ ID NO:143 and two polypeptidechains comprising the sequence of SEQ ID NO:140.

II.H.5 DhCpmFc(−)(N297G)(Y349C)-GDF15(N3D):DhCpmFc(+)(N297G)(S354C)

The designation“DhCpmFc(−)(N297G)(Y349C)-GDF15(N3D):DhCpmFc(+)(N297G)(S354C)” in theinstant disclosure refers to a heterodimer comprising (i) a firstpolypeptide chain comprising a GDF15(N3D) polypeptide, the N-terminus ofwhich is linked directly to the C-terminus of a DhCpmFc(−)(N297G)(Y349C)domain, and (ii) a second polypeptide chain comprising aDhCpmFc(+)(N297G)(S345C) domain, The cysteine clamp mutations allow thefirst and second polypeptide chains to be linked via an interchaindisulfide bond between C349 of the first polypeptide chain and C354 ofthe second polypeptide chain.

In certain embodiments, a tetramer is provided, comprising a dimer oftwo DhCpmFc(−)(N297G)(Y349C)-GDF15(N3D):DhCpmFc(+)(N297G)(S354C)heterodimers in which the two first polypeptide chains of eachheterodimer are linked via an interchain disulfide bond between theirrespective GDF15 regions.

More particularly, in a specific embodiment, the tetramer comprises:

(a) two DhCpmFc(+)(N297G)(S354C) domains (one each heterodimer)comprising the sequence of SEQ ID NO:288,

(b) two DhCpmFc(−)(N297G)(Y349C) domains comprising the sequence of SEQID NO:141, and

(c) two GDF15(N3D) polypeptides (one each heterodimer) comprising thesequence of SEQ ID NO:52.

In a preferred embodiment, the first polypeptide chain comprises theamino acid sequence:

(SEQ ID NO: 146) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGARDGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 145) gcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacgggagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtgcaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtgcgcgcgacggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcata.

In a preferred embodiment, the second polypeptide chain comprises theamino acid sequence of SEQ ID NO:140, which is encoded by the nucleicacid sequence of SEQ ID NO:144.

As discussed above, a tetramer is provided comprising two polypeptidechains comprising the sequence of SEQ ID NO:146 and two polypeptidechains comprising the sequence of SEQ ID NO:140.

II.H.6 DhCpmFc(−)(N297G)(L351C)-GDF15(Ndel3):DhCpmFc(+)(N297G)(L351C)

The designation“DhCpmFc(−)(N297G)(L351C)-GDF15(Ndel3):DhCpmFc(+)(N297G)(L351C)” in theinstant disclosure refers to a heterodimer comprising (i) a firstpolypeptide chain comprising a GDF15(Ndel3) polypeptide, the N-terminusof which is linked directly to the C-terminus of aDhCpmFc(−)(N297G)(L351C) domain, and (ii) a second polypeptide chaincomprising a DhCpmFc(+)(N297G)(L351C) domain. The cysteine clampmutations allow the first and second polypeptide chains to be linked viaan interchain disulfide bond between C351 of the first polypeptide chainand C351 of the second polypeptide chain.

In certain embodiments, a tetramer is provided, comprising a dimer oftwo DhCpmFc(−)(N297G)(L351C)-GDF15(Ndel3):DhCpmFc(+)(N297G)(L351C)heterodimers in which the two first polypeptide chains of eachheterodimer are linked via an interchain disulfide bond between theirrespective GDF15 regions.

More particularly, in a specific embodiment, the tetramer comprises:

(a) two DhCpmFc(+)(N297G)(L351C) domains (one each heterodimer)comprising the sequence:

(SEQ ID NO: 289) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTCPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPG,

(b) two DhCpmFc(−)(N297G)(L351C) domains (one each heterodimer)comprising the sequence:

(SEQ ID NO: 148) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTCPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPG,and

(c) two GDF15(Ndel3) polypeptides (one each heterodimer) comprising thesequence of SEQ ID NO:55.

In a preferred embodiment, the first polypeptide chain comprises theamino acid sequence:

(SEQ ID NO: 150) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTCPPSRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 149) gcgccggaactgctgggcggcccgagcgtgtttctgtttccgccgaaaccgaaagataccctgatgattagccgcaccccggaagtgacctgcgtggtggtggatgtgagccatgaagatccggaagtgaaatttaactggtatgtggatggcgtggaagtgcataacgcgaaaaccaaaccgcgcgaagaacagtatggcagcacctatcgcgtggtgagcgtgctgaccgtgctgcatcaggattggctgaacggcaaagaatataaatgcaaagtgagcaacaaagcgctgccggcgccgattgaaaaaaccattagcaaagcgaaaggccagccgcgcgaaccgcaggtgtatacctgcccgccgagccgcaaagaaatgaccaaaaaccaggtgagcctgacctgcctggtgaaaggcttttatccgagcgatattgcggtggaatgggaaagcaacggccagccggaaaacaactataaaaccaccccgccggtgctgaaaagcgatggcagcttttttctgtatagcaaactgaccgtggataaaagccgctggcagcagggcaacgtgtttagctgcagcgtgatgcatgaagcgctgcataaccattatacccagaaaagcctgagcctgagcccgggcgcgcgcaacggcgatcattgcccgctgggcccgggccgctgctgccgcctgcataccgtgcgcgcgagcctggaagatctgggctgggcggattgggtgctgagcccgcgcgaagtgcaggtgaccatgtgcattggcgcgtgcccgagccagtttcgcgcggcgaacatgcatgcgcagattaaaaccagcctgcatcgcctgaaaccggataccgtgccggcgccgtgctgcgtgccggcgagctataacccgatggtgctgattcagaaaaccgataccggcgtgagcctgcagacctatgatgatctgctggcgaaagattgccattgcatt.

In a preferred embodiment, the second polypeptide chain comprises theamino acid sequence:

(SEQ ID NO: 147) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTCPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 151) gcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacgggagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacacctgtcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaa a.

As discussed above, a tetramer is provided comprising two polypeptidechains comprising the sequence of SEQ ID NO:150 and two polypeptidechains comprising the sequence of SEQ ID NO:147.

II.H.7 DhCpmFc(−)(N297G)(L351C)-GDF15(N3D):DhCpmFc(+)(N297G)(L351C)

The designation“DhCpmFc(−)(N297G)(L351C)-GDF15(N3D):DhCpmFc(+)(N297G)(L351C)” in theinstant disclosure refers to a heterodimer comprising (i) a firstpolypeptide chain comprising a GDF15(N3D) polypeptide, the N-terminus ofwhich is linked directly to the C-terminus of a DhCpmFc(−)(N297G)(L351C)domain by a peptide bond and (ii) a second polypeptide chain comprisinga DhCpmFc(+)(N297G)(L351C) domain. The cysteine clamp mutations allowthe first and second polypeptide chains to be linked via an interchaindisulfide bond between C351 of the first polypeptide chain and C351 ofthe second polypeptide chain.

In certain embodiments, a tetramer is provided, comprising a dimer oftwo DhCpmFc(−)(N297G)(L351C)-GDF15(N3D):DhCpmFc(+)(N297G)(L351C)heterodimers in which the two first polypeptide chains of eachheterodimer are linked via an interchain disulfide bond between theirrespective GDF15 regions.

More particularly, in a specific embodiment, the tetramer comprises:

(a) two DhCpmFc(+)(N297G)(L351C) domains (one each heterodimer)comprising the sequence of SEQ ID NO:289,

(b) two DhCpmFc(−)(N297G)(L351C) domains (one each heterodimer)comprising the sequence of SEQ ID NO:148, and

(c) two GDF15(N3D) polypeptides (one each heterodimer) comprising thesequence of SEQ ID NO:52.

In a preferred embodiment, the first polypeptide chain comprises theamino acid sequence:

(SEQ ID NO: 153) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTCPPSRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGARDGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 152) gcgccggaactgctgggcggcccgagcgtgtttctgtttccgccgaaaccgaaagataccctgatgattagccgcaccccggaagtgacctgcgtggtggtggatgtgagccatgaagatccggaagtgaaatttaactggtatgtggatggcgtggaagtgcataacgcgaaaaccaaaccgcgcgaagaacagtatggcagcacctatcgcgtggtgagcgtgctgaccgtgctgcatcaggattggctgaacggcaaagaatataaatgcaaagtgagcaacaaagcgctgccggcgccgattgaaaaaaccattagcaaagcgaaaggccagccgcgcgaaccgcaggtgtatacctgcccgccgagccgcaaagaaatgaccaaaaaccaggtgagcctgacctgcctggtgaaaggcttttatccgagcgatattgcggtggaatgggaaagcaacggccagccggaaaacaactataaaaccaccccgccggtgctgaaaagcgatggcagcttttttctgtatagcaaactgaccgtggataaaagccgctggcagcagggcaacgtgtttagctgcagcgtgatgcatgaagcgctgcataaccattatacccagaaaagcctgagcctgagcccgggcgcgcgcgatggcgatcattgcccgctgggcccgggccgctgctgccgcctgcataccgtgcgcgcgagcctggaagatctgggctgggcggattgggtgctgagcccgcgcgaagtgcaggtgaccatgtgcattggcgcgtgcccgagccagtttcgcgcggcgaacatgcatgcgcagattaaaaccagcctgcatcgcctgaaaccggataccgtgccggcgccgtgctgcgtgccggcgagctataacccgatggtgctgattcagaaaaccgataccggcgtgagcctgcagacctatgatgatctgctggcgaaagattgccattgcatt.

In a preferred embodiment, the second polypeptide chain comprises theamino acid sequence of SEQ ID NO:147, which is encoded by the nucleicacid sequence of SEQ ID NO:151.

As discussed above, a tetramer is provided comprising two polypeptidechains comprising the sequence of SEQ ID NO:153 and two polypeptidechains comprising the sequence of SEQ ID NO:147.

II.H.8 DhCpmFc(−)(N297G)(A287C)-GDF15(Ndel3):DhCpmFc(+)(N297G)(L306C)

The designation“DhCpmFc(−)(N297G)(A287C)-GDF15(Ndel3):DhCpmFc(+)(N297G)(L306C)” in theinstant disclosure refers to a heterodimer comprising (i) a firstpolypeptide chain comprising a GDF15(Ndel3) polypeptide, the N-terminusof which is linked directly to the C-terminus of aDhCpmFc(−)(N297G)(A287C) domain, and (ii) a second polypeptide chaincomprising a DhCpmFc(+)(N297G)(L306C) domain.

In certain embodiments, a tetramer is provided, comprising a dimer oftwo DhCpmFc(−)(N297G)(A287C)-GDF15(Ndel3):DhCpmFc(+)(N297G)(L306C)heterodimers in which the two first polypeptide chains of eachheterodimer are linked via an interchain disulfide bond between theirrespective GDF15 regions.

More particularly, in a specific embodiment, the tetramer comprises:

(a) two DhCpmFc(+)(N297G)(L306C) domains (one each heterodimer)comprising the sequence:

(SEQ ID NO: 290) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNCKTKPREEQYGSTYRVVSVCTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPG,

(b) two DhCpmFc(−)(N297G)(A287C) domains (one each heterodimer)comprising the sequence:

(SEQ ID NO: 268) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNCKTKPREEQYGSTYRVVSVCTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPG,and

(c) two GDF15(Ndel3) polypeptides (one each heterodimer) comprising thesequence of SEQ ID NO:55.

In a preferred embodiment, the first polypeptide chain comprises theamino acid sequence:

(SEQ ID NO: 269) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNCKTKPREEQYGSTYRVVSVCTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 270) gcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataattgcaagacaaagccgcgggaggagcagtacggcagcacgtaccgtgtggtcagcgtctgcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcata.

In a preferred embodiment, the second polypeptide chain comprises theamino acid sequence:

(SEQ ID NO: 267) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNCKTKPREEQYGSTYRVVSVCTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 271) gcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataattgcaagacaaagccgcgggaggagcagtacggcagcacgtaccgtgtggtcagcgtctgcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccggaaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctgaagtccgacggctccttcttcctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaa a.

As discussed above, a tetramer is provided comprising two polypeptidechains comprising the sequence of SEQ ID NO:269 and two polypeptidechains comprising the sequence of SEQ ID NO:267.

II.H.9 DhCpmFc(−)(N297G)(A287C)-GDF15(N3D):DhCpmFc(+)(N297G)(L306C)

The designation“DhCpmFc(−)(N297G)(A287C)-GDF15(N3D):DhCpmFc(+)(N297G)(L306C)” in theinstant disclosure refers to a heterodimer comprising (i) a firstpolypeptide chain comprising a GDF15(N3D) polypeptide, the N-terminus ofwhich is linked directly to the C-terminus of a DhCpmFc(−)(N297G)(A287C)domain, and (ii) a second polypeptide chain comprising aDhCpmFc(+)(N297G)(L306C) domain.

In certain embodiments, a tetramer is provided, comprising a dimer oftwo DhCpmFc(−)(N297G)(A287C)-GDF15(N3D):DhCpmFc(+)(N297G)(L306C)heterodimers in which the two first polypeptide chains of eachheterodimer are linked via an interchain disulfide bond between theirrespective GDF15 regions.

More particularly, in a specific embodiment, the tetramer comprises:

(a) two DhCpmFc(+)(N297G)(L306C) domains (one each heterodimer)comprising the sequence of SEQ ID NO:290,

(b) two DhCpmFc(−)(N297G)(A287C) domains (one each heterodimer)comprising the sequence of SEQ ID NO:268, and

(c) two GDF15(N3D) polypeptides (one each heterodimer) comprising thesequence of SEQ ID NO:52.

In a preferred embodiment, the first polypeptide chain comprises theamino acid sequence:

(SEQ ID NO: 272) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNCKTKPREEQYGSTYRVVSVCTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGARDGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 273) gcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataattgcaagacaaagccgcgggaggagcagtacggcagcacgtaccgtgtggtcagcgtctgcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtgcgcgcgacggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcata.

In a preferred embodiment, the second polypeptide chain comprises theamino acid sequence of SEQ ID NO:267, which is encoded by the nucleicacid sequence of SEQ ID NO:271.

As discussed above, a tetramer is provided comprising two polypeptidechains comprising the sequence of SEQ ID NO:272 and two polypeptidechains comprising the sequence of SEQ ID NO:267.

II.H.10DhCpmFc(−)(N297G)(A287C)(Y349C)-GDF15(Ndel3):DhCpmFc(+)(N297G)(L306C)(S354C)

The designation“DhCpmFc(−)(N297G)(A287C)(Y349C)-GDF15(Ndel3):DhCpmFc(+)(N297G)(L306C)(S354C)”in the instant disclosure refers to a heterodimer comprising (i) a firstpolypeptide chain comprising a GDF15(Ndel3) polypeptide, the N-terminusof which is linked directly to the C-terminus of aDhCpmFc(−)(N297G)(A287C)(Y349C) domain, and (ii) a second polypeptidechain comprising a DhCpmFc(+)(N297G)(L306C)(S354C) domain.

In certain embodiments, a tetramer is provided, comprising a dimer oftwoDhCpmFc(−)(N297G)(A287C)(Y349C)-GDF15(Ndel3):DhCpmFc(+)(N297G)(L306C)(S354C)heterodimers in which the two first polypeptide chains of eachheterodimer are linked via an interchain disulfide bond between theirrespective GDF15 regions.

More particularly, in a specific embodiment, the tetramer comprises:

(a) two DhCpmFc(+)(N297G)(L306C)(Y349C) domains (one each heterodimer)comprising the sequence:

(SEQ ID NO: 291) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNCKTKPREEQYGSTYRVVSVCTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPG,

(b) two DhCpmFc(−)(N297G)(A287C)(S354C) domains (one each heterodimer)comprising the sequence:

(SEQ ID NO: 275) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNCKTKPREEQYGSTYRVVSVCTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPG,and

(c) two GDF15(Ndel3) polypeptides (one each heterodimer) comprising thesequence of SEQ ID NO:55.

In a preferred embodiment, the first polypeptide chain comprises theamino acid sequence:

(SEQ ID NO: 276) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNCKTKPREEQYGSTYRVVSVCTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 277) gcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataattgcaagacaaagccgcgggaggagcagtacggcagcacgtaccgtgtggtcagcgtctgcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtgcaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcata.

In a preferred embodiment, the second polypeptide chain comprises theamino acid sequence:

(SEQ ID NO: 274) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNCKTKPREEQYGSTYRVVSVCTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 278) gcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataattgcaagacaaagccgcgggaggagcagtacggcagcacgtaccgtgtggtcagcgtctgcaccgtgctccaccaggactggcttaatgggaaggaatacaagtgtaaggtgtccaacaaggccctccccgctcccatcgaaaagaccatctcaaaggcaaaggggcaaccaagggaacctcaagtgtacaccctgcctccgtgcaggaaggagatgaccaagaaccaggtcagcctgacttgtctcgtgaagggcttctatcccagcgatattgctgtggaatgggagtcaaatggccagcccgagaataactacaaaactaccccacccgtgctgaaatctgatgggtccttcttcctttactccaagctgaccgtggacaagagccgctggcaacaaggcaatgtctttagctgctcagtgatgcatgaggctctccataatcactacactcagaagtcactgtccctgtctccgggtaa a.

As discussed above, a tetramer is provided comprising two polypeptidechains comprising the sequence of SEQ ID NO:276 and two polypeptidechains comprising the sequence of SEQ ID NO:274.

II.H.11DhCpmFc(−)(N297G)(A287C)(Y349C)-GDF15(N3D):DhCpmFc(+)(N297G)(L306C)(S354C)

The designation“DhCpmFc(−)(N297G)(A287C)(Y349C)-GDF15(N3D):DhCpmFc(+)(N297G)(L306C)(S354C)”in the instant disclosure refers to a heterodimer comprising (i) a firstpolypeptide chain comprising a GDF15(N3D) polypeptide, the N-terminus ofwhich is linked directly to the C-terminus of aDhCpmFc(−)(N297G)(A287C)(Y349C) domain, and (ii) a second polypeptidechain comprising a DhCpmFc(+)(N297G)(L306C)(S354C) domain.

In certain embodiments, a tetramer is provided, comprising a dimer oftwoDhCpmFc(−)(N297G)(A287C)(Y349C)-GDF15(N3D):DhCpmFc(+)(N297G)(L306C)(S354C)heterodimers in which the two first polypeptide chains of eachheterodimer are linked via an interchain disulfide bond between theirrespective GDF15 regions.

More particularly, in a specific embodiment, the tetramer comprises:

(a) two DhCpmFc(+)(N297G)(L306C)(Y349C) domains (one each heterodimer)comprising the sequence of SEQ ID NO:291,

(b) two DhCpmFc(−)(N297G)(A287C)(S354C) domains (one each heterodimer)comprising the sequence of SEQ ID NO:275, and

(c) two GDF15(N3D) polypeptides (one each heterodimer) comprising thesequence of SEQ ID NO:52.

In a preferred embodiment, the first polypeptide chain comprises theamino acid sequence:

(SEQ ID NO: 279) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNCKTKPREEQYGSTYRVVSVCTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGARDGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 280) gcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataattgcaagacaaagccgcgggaggagcagtacggcagcacgtaccgtgtggtcagcgtctgcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtgcaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtgcgcgcgacggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcata.

In a preferred embodiment, the second polypeptide chain comprises theamino acid sequence of SEQ ID NO:274, which is encoded by the nucleicacid sequence of SEQ ID NO:278.

As discussed above, a tetramer is provided comprising two polypeptidechains comprising the sequence of SEQ ID NO:279 and two polypeptidechains comprising the sequence of SEQ ID NO:274.

II.H.12 Dh2CpmFc(−)-GDF15(Ndel3):Dh2CpmFc(+)

The designation “Dh2CpmFc(−)-GDF15(Ndel3):Dh2CpmFc(+)” in the instantdisclosure refers to a heterodimer comprising (i) a first polypeptidechain comprising a GDF15(Ndel3) polypeptide, the N-terminus of which islinked directly to the C-terminus of a Dh2CpmFc(−) domain, and (ii) asecond polypeptide chain comprising a Dh2CpmFc(+) domain.

In certain embodiments, a tetramer is provided, comprising a dimer oftwo Dh2CpmFc(−)-GDF15(Ndel3):Dh2CpmFc(+) heterodimers in which the twofirst polypeptide chains of each heterodimer are linked via aninterchain disulfide bond between their respective GDF15 regions.

More particularly, in a specific embodiment, the tetramer comprises:

(a) two Dh2CpmFc(+) domains (one each heterodimer) comprising thesequence:

(SEQ ID NO: 292) PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG,

(b) two Dh2CpmFc(−) domains (one each heterodimer) comprising thesequence:

(SEQ ID NO: 155) PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG,and

(c) two GDF15(Ndel3) polypeptides (one each heterodimer) comprising thesequence of SEQ ID NO:55.

In a preferred embodiment, the first polypeptide chain comprises theamino acid sequence:

(SEQ ID NO: 157) PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDT GVSLQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 156) ccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactg cata.

In a preferred embodiment, the second polypeptide chain comprises theamino acid sequence:

(SEQ ID NO: 154) PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 158) ccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccggaaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctgaagtccgacggctccttcttcctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga

As discussed above, a tetramer is provided comprising two polypeptidechains comprising the sequence of SEQ ID NO:157 and two polypeptidechains comprising the sequence of SEQ ID NO:154.

II.H.13 Dh2CpmFc(−)-GDF15(N3D):Dh2CpmFc(+)

The designation “Dh2CpmFc(−)-GDF15(N3D):Dh2CpmFc(+)” in the instantdisclosure refers to a heterodimer comprising (i) a first polypeptidechain comprising a GDF15(N3D) polypeptide, the N-terminus of which islinked directly to the C-terminus of a Dh2CpmFc(−) domain by a peptidebond, and (ii) a second polypeptide chain comprising a Dh2CpmFc(+)domain.

In certain embodiments, a tetramer is provided, comprising a dimer oftwo Dh2CpmFc(−)-GDF15(N3D):DhCpmFc(+) heterodimers in which the twofirst polypeptide chains of each heterodimer are linked via aninterchain disulfide bond between their respective GDF15 regions.

More particularly, in a specific embodiment, the tetramer comprises:

(a) two Dh2CpmFc(+) domains (one each heterodimer) comprising thesequence of SEQ ID NO:292,

(b) two Dh2CpmFc(−) domains (one each heterodimer) comprising thesequence of SEQ ID NO:155, and

(c) two GDF15(N3D) polypeptides (one each heterodimer) comprising thesequence of SEQ ID NO:52.

In a preferred embodiment, the first polypeptide chain comprises theamino acid sequence:

(SEQ ID NO: 160) PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGARDGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 159) ccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtgcgcgcgacggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaaga ctgccactgcata.

In a preferred embodiment, the second polypeptide chain comprises theamino acid sequence of SEQ ID NO:154, which is encoded by the nucleicacid sequence of SEQ ID NO:158.

As discussed above, a tetramer is provided comprising two polypeptidechains comprising the sequence of SEQ ID NO:160 and two polypeptidechains comprising the sequence of SEQ ID NO:154.

II.H.14 Dh2CpmFc(−)(Y349C)-GDF15(Ndel3):Dh2CpmFc(+)(S354C)

The designation “Dh2CpmFc(−)(Y349C)-GDF15(Ndel3):Dh2CpmFc(+)(S354C)” inthe instant disclosure refers to a heterodimer comprising (i) a firstpolypeptide chain comprising a GDF15(Ndel3) polypeptide, the N-terminusof which is linked directly to the C-terminus of a Dh2CpmFc(−)(Y349C)domain, and (ii) a second polypeptide chain comprising aDh2CpmFc(+)(S345C) domain. The cysteine clamp mutations allow the firstand second polypeptide chains to be linked via an interchain disulfidebond between C349 of the first polypeptide chain and C354 of the secondpolypeptide chain.

In certain embodiments, a tetramer is provided, comprising a dimer oftwo Dh2CpmFc(−)(Y349C)-GDF15(Ndel3):Dh2CpmFc(+)(S354C) heterodimers inwhich the two first polypeptide chains of each heterodimer are linkedvia an interchain disulfide bond between their respective GDF15 regions.

More particularly, in a specific embodiment, the tetramer comprises:

(a) two Dh2CpmFc(+)(S354C) domains (one each heterodimer) comprising thesequence:

(SEQ ID NO: 293) PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG,

(b) two Dh2CpmFc(−)(Y349C) domains (one each heterodimer) comprising thesequence:

(SEQ ID NO: 162) PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG,and

(c) two GDF15(Ndel3) polypeptides (one each heterodimer) comprising thesequence of SEQ ID NO:55.

In a preferred embodiment, the first polypeptide chain comprises theamino acid sequence:

(SEQ ID NO: 164) PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDT GVSLQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 163) ccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtgcaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactg cata.

In a preferred embodiment, the second polypeptide chain comprises theamino acid sequence:

(SEQ ID NO: 161) PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 165) ccatccgtgttcctgtttcctccaaagccgaaggacaccctgatgatctcaagaactccggaagtgacttgcgtcgtcgtggacgtgtcacatgaggatccagaggtcaagttcaattggtatgtggacggagtggaagtgcataacgccaagaccaaaccccgcgaagaacagtacaatagcacctaccgcgtggtgagcgtccttactgtgctccaccaggactggcttaatgggaaggaatacaagtgtaaggtgtccaacaaggccctccccgctcccatcgaaaagaccatctcaaaggcaaaggggcaaccaagggaacctcaagtgtacaccctgcctccgtgcaggaaggagatgaccaagaaccaggtcagcctgacttgtctcgtgaagggcttctatcccagcgatattgctgtggaatgggagtcaaatggccagcccgagaataactacaaaactaccccacccgtgctgaaatctgatgggtccttcttcctttactccaagctgaccgtggacaagagccgctggcaacaaggcaatgtctttagctgctcagtgatgcatgaggctctccataatcactacactcagaagtcactgtccctgtctccgggtaaa.

As discussed above, a tetramer is provided comprising two polypeptidechains comprising the sequence of SEQ ID NO:164 and two polypeptidechains comprising the sequence of SEQ ID NO:161.

II.H.15 Dh2CpmFc(−)(Y349C)-GDF15(N3D):Dh2CpmFc(+)(S354C)

The designation “Dh2CpmFc(−)(Y349C)-GDF15(N3D):Dh2CpmFc(+)(S354C)” inthe instant disclosure refers to a heterodimer comprising (i) a firstpolypeptide chain comprising a GDF15(N3D) polypeptide, the N-terminus ofwhich is linked directly to the C-terminus of a Dh2CpmFc(−)(Y349C)domain, and (ii) a second polypeptide chain comprising aDh2CpmFc(+)(S345C) domain. The cysteine clamp mutations allow the firstand second polypeptide chains to be linked via an interchain disulfidebond between C349 of the first polypeptide chain and C354 of the secondpolypeptide chain.

In certain embodiments, a tetramer is provided, comprising a dimer oftwo Dh2CpmFc(−)(Y349C)-GDF15(N3D):Dh2CpmFc(+)(S354C) heterodimers inwhich two first polypeptide chains of each heterodimer are linked via aninterchain disulfide bond between their respective GDF15 regions.

More particularly, in a specific embodiment, the tetramer comprises:

(a) two Dh2CpmFc(+)(S354C) domains (one each heterodimer) comprising thesequence of SEQ ID NO:293,

(b) two Dh2CpmFc(−)(Y349C) domains (one each heterodimer) comprising thesequence of SEQ ID NO:162, and

(c) two GDF15(N3D) polypeptides (one each heterodimer) comprising thesequence of SEQ ID NO:52.

In a preferred embodiment, the first polypeptide chain comprises theamino acid sequence:

(SEQ ID NO: 167) PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGARDGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 166) ccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtgcaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtgcgcgcgacggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaaga ctgccactgcata.

In a preferred embodiment, the second monomer comprises the amino acidsequence of SEQ ID NO:161, which is encoded by the nucleic acid sequenceof SEQ ID NO:165.

As discussed above, a tetramer is provided comprising two polypeptidechains comprising the sequence of SEQ ID NO:167 and two polypeptidechains comprising the sequence of SEQ ID NO:161.

II.H.16 CpmFc(−)(N297G)-GDF15(Ndel3):CpmFc(+)(N297G)

The designation “CpmFc(−)(N297G)-GDF15(Ndel3):DhCpmFc(+)(N297G)” in theinstant disclosure refers to a heterodimer comprising (i) a firstpolypeptide chain comprising a GDF15(Ndel3) polypeptide, the N-terminusof which is linked directly to the C-terminus of a CpmFc(−)(N297G)domain, and (ii) a second polypeptide chain comprising a CpmFc(+)(N297G)domain.

In certain embodiments, a tetramer is provided, comprising a dimer oftwo CpmFc(−)(N297G)-GDF15(Ndel3):CpmFc(+)(N297G) heterodimers in whichthe two first polypeptide chains of each heterodimer are linked via aninterchain disulfide bond between their respective GDF15 regions.

More particularly, in a specific embodiment, the tetramer comprises:

(a) two CpmFc(+)(N297G) domains (one each heterodimer) comprising thesequence (part of the hinge region in parentheses):

(SEQ ID NO: 294) (DKTHTCPPCP)APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG,

(b) two CpmFc(−)(N297G) domains (one each heterodimer) comprising thesequence (part of the hinge region in parentheses):

(SEQ ID NO: 169) (DKTHTCPPCP)APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG,and

(c) two GDF15(Ndel3) polypeptides (one each heterodimer) comprising thesequence of SEQ ID NO:55.

In a preferred embodiment, the first polypeptide chain comprises theamino acid sequence:

(SEQ ID NO: 171) (DKTHTCPPCP)APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 170) gacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacggcagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagac tgccactgcata.

In a preferred embodiment, the second polypeptide chain comprises theamino acid sequence:

(SEQ ID NO: 168) (DKTHTCPPCP)APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 172) gacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacggcagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccggaaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctgaagtccgacggctccttcttcctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa.

As discussed above, a tetramer is provided comprising two polypeptidechains comprising the sequence of SEQ ID NO:171 and two polypeptidechains comprising the sequence of SEQ ID NO:168.

II.H.17 CpmFc(−)(N297G)-GDF15(N3D):CpmFc(+)(N297G)

The designation “CpmFc(−)(N297G)-GDF15(N3D):DhCpmFc(+)(N297G)” in theinstant disclosure refers to a heterodimer comprising (i) a firstpolypeptide chain comprising a GDF15(N3D) polypeptide, the N-terminus ofwhich is linked directly to the C-terminus of a CpmFc(−)(N297G) domain,and (ii) a second polypeptide chain comprising a CpmFc(+)(N297G) domain.

In certain embodiments, a tetramer is provided, comprising a dimer oftwo CpmFc(−)(N297G)-GDF15(N3D):CpmFc(+)(N297G) heterodimers in which thetwo first polypeptide chains of each heterodimer are linked via aninterchain disulfide bond between their respective GDF15 regions.

More particularly, in a specific embodiment, the tetramer comprises:

(a) two CpmFc(+)(N297G) domains (one each heterodimer) comprising thesequence of SEQ ID NO:294,

(b) two CpmFc(−)(N297G) domains (one each heterodimer) comprising thesequence of SEQ ID NO:169, and

(c) two GDF15(N3D) polypeptides (one each heterodimer) comprising thesequence of SEQ ID NO:52.

In a preferred embodiment, the first polypeptide chain comprises theamino acid sequence:

(SEQ ID NO: 174) (DKTHTCPPCP)APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGARDGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 173) gacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacggcagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtgcgcgcgacggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaag actgccactgcata.

In a preferred embodiment, the second polypeptide chain comprises theamino acid sequence of SEQ ID NO:168, which is encoded by the nucleicacid sequence comprising the sequence of SEQ ID NO:172.

As discussed above, a tetramer is provided comprising two polypeptidechains comprising the sequence of SEQ ID NO:174 and two polypeptidechains comprising the sequence of SEQ ID NO:168.

II.H.18 Dh2CpmFc(−)(N297G)-GDF15(Ndel3):Dh2CpmFc(+)(N297G)

The designation “Dh2CpmFc(−)(N297G)-GDF15(Ndel3):Dh2CpmFc(+)(N297G)” inthe instant disclosure refers to a heterodimer comprising (i) a firstpolypeptide chain comprising a GDF15(Ndel3) polypeptide, the N-terminusof which is linked directly to the C-terminus of a Dh2CpmFc(−)(N297G)domain, and (ii) a second polypeptide chain comprising aDh2CpmFc(+)(N297G) domain.

In certain embodiments, a tetramer is provided, comprising a dimer oftwo Dh2CpmFc(−)(N297G)-GDF15(Ndel3):Dh2CpmFc(+)(N297G) heterodimers inwhich the two first polypeptide chains of each heterodimer are linkedvia an interchain disulfide bond between their respective GDF15 regions.

More particularly, in a specific embodiment, the tetramer comprises:

(a) two Dh2CpmFc(+)(N297G) domains (one each heterodimer) comprising thesequence:

(SEQ ID NO: 295) PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG,

(b) two Dh2CpmFc(−)(N297G) domains (one each heterodimer) comprising thesequence:

(SEQ ID NO: 176) PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG,and

(c) two GDF15(Ndel3) polypeptides (one each heterodimer) comprising thesequence of SEQ ID NO:55.

In a preferred embodiment, the first polypeptide chain comprises theamino acid sequence:

(SEQ ID NO: 178) PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDT GVSLQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 177) ccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacgggagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactg cata.

In a preferred embodiment, the second polypeptide chain comprises theamino acid sequence:

(SEQ ID NO: 175) PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 179) ccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacgggagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccggaaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctgaagtccgacggctccttcttcctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga.

As discussed above, a tetramer is provided comprising two polypeptidechains comprising the sequence of SEQ ID NO:178 and two polypeptidechains comprising the sequence of SEQ ID NO:175.

II.H.19 Dh2CpmFc(−)(N297G)-GDF15(N3D):Dh2CpmFc(+)(N297G)

The designation “Dh2CpmFc(−)(N297G)-GDF15(N3D):DhCpmFc(+)(N297G)” in theinstant disclosure refers to a heterodimer comprising (i) a firstpolypeptide chain comprising a GDF15(N3D) polypeptide, the N-terminus ofwhich is linked directly to the C-terminus of a Dh2CpmFc(−)(N297G)domain, and (ii) a second polypeptide chain comprising aDh2CpmFc(+)(N297G) domain.

In certain embodiments, a tetramer is provided, comprising a dimer oftwo Dh2CpmFc(−)(N297G)-GDF15(N3D):Dh2CpmFc(+)(N297G) heterodimers inwhich the two first polypeptide chains of each heterodimer are linkedvia an interchain disulfide bond between their respective GDF15 regions.

More particularly, in a specific embodiment, the tetramer comprises:

(a) two Dh2CpmFc(+)(N297G) domains (one each heterodimer) comprising thesequence of SEQ ID NO:295,

(b) two Dh2CpmFc(−)(N297G) domains (one each heterodimer) comprising thesequence of SEQ ID NO:176, and

(c) two GDF15(N3D) polypeptides (one each heterodimer) comprising thesequence of SEQ ID NO:52.

In a preferred embodiment, the first polypeptide chain comprises theamino acid sequence:

(SEQ ID NO: 181) PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGARDGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 180) ccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacgggagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtgcgcgcgacggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaaga ctgccactgcata.

In a preferred embodiment, the second polypeptide chain comprises theamino acid sequence of SEQ ID NO:175, which is encoded by the nucleicacid sequence of SEQ ID NO:179.

As discussed above, a tetramer is provided comprising two polypeptidechains comprising the sequence of SEQ ID NO:181 and two polypeptidechains comprising the sequence of SEQ ID NO:175.

II.H.20 Dh2CpmFc(−)(N297G)(Y349C)-GDF15(Ndel3):Dh2CpmFc(+)(N297G)(S354C)

The designation“Dh2CpmFc(−)(N297G)(Y349C)-GDF15(Ndel3):Dh2CpmFc(+)(N297G)(S354C)” inthe instant disclosure refers to a heterodimer comprising (i) a firstpolypeptide chain comprising a GDF15(Ndel3) polypeptide, the N-terminusof which is linked directly to the C-terminus of aDh2CpmFc(−)(N297G)(Y349C) polypeptide, and (ii) a second polypeptidechain comprising a Dh2CpmFc(+)(N297G)(S345C) domain. The cysteine clampmutations allow the first and second polypeptide chains to be linked viaan interchain disulfide bond between C349 of the first polypeptide chainand C354 of the second polypeptide chain.

In certain embodiments, a tetramer is provided, comprising a dimer oftwo Dh2CpmFc(−)(N297G)(Y349C)-GDF15(Ndel3):Dh2CpmFc(+)(N297G)(S354C)heterodimers in which the two first polypeptide chains of eachheterodimer are linked via an interchain disulfide bond between theirrespective GDF15 regions.

More particularly, in a specific embodiment, the tetramer comprises:

(a) two Dh2CpmFc(+)(N297G)(S354C) domains (one each heterodimer)comprising the sequence:

(SEQ ID NO: 296) PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG,

(b) two Dh2CpmFc(−)(N297G)(Y349C) domains (one each heterodimer)comprising the sequence:

(SEQ ID NO: 183) PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG,and

(c) two GDF15(Ndel3) polypeptides (one each heterodimer) comprising thesequence of SEQ ID NO:55.

In a preferred embodiment, the first polypeptide chain comprises theamino acid sequence:

(SEQ ID NO: 185) PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDT GVSLQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 184) ccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacggcagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtgcaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactg cata.

In a preferred embodiment, the second polypeptide chain comprises theamino acid sequence:

(SEQ ID NO: 182) PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 186) ccatccgtgttcctgtttcctccaaagccgaaggacaccctgatgatctcaagaactccggaagtgacttgcgtcgtcgtggacgtgtcacatgaggatccagaggtcaagttcaattggtatgtggacggagtggaagtgcataacgccaagaccaaaccccgcgaagaacagtacgggagcacctaccgcgtggtgagcgtccttactgtgctccaccaggactggcttaatgggaaggaatacaagtgtaaggtgtccaacaaggccctccccgctcccatcgaaaagaccatctcaaaggcaaaggggcaaccaagggaacctcaagtgtacaccctgcctccgtgcaggaaggagatgaccaagaaccaggtcagcctgacttgtctcgtgaagggcttctatcccagcgatattgctgtggaatgggagtcaaatggccagcccgagaataactacaaaactaccccacccgtgctgaaatctgatgggtccttcttcctttactccaagctgaccgtggacaagagccgctggcaacaaggcaatgtctttagctgctcagtgatgcatgaggctctccataatcactacactcagaagtcactgtccctgtctccgggtaaa.

As discussed above, a tetramer is provided comprising two polypeptidechains comprising the sequence of SEQ ID NO:185 and two polypeptidechains comprising the sequence of SEQ ID NO:182.

II.H.21 Dh2CpmFc(−)(N297G)(Y349C)-GDF15(N3D):Dh2CpmFc(+)(N297G)(S354C)

The designation“Dh2CpmFc(−)(N297G)(Y349C)-GDF15(N3D):Dh2CpmFc(+)(N297G)(S354C)” in theinstant disclosure refers to a heterodimer comprising (i) a firstpolypeptide chain comprising a GDF15(N3D) polypeptide, the N-terminus ofwhich is linked directly to the C-terminus of aDh2CpmFc(−)(N297G)(Y349C) domain, and (ii) a second polypeptide chaincomprising a Dh2CpmFc(+)(N297G)(S345C) domain The cysteine clampmutations allow the first and second polypeptide chains to be linked viaan interchain disulfide bond between C349 of the first polypeptide andC354 of the second polypeptide.

In certain embodiments, a tetramer is provided, comprising a dimer oftwo Dh2CpmFc(−)(N297G)(Y349C)-GDF15(N3D):Dh2CpmFc(+)(N297G)(S354C)heterodimers in which the two first polypeptide chains of eachheterodimer are linked via an interchain disulfide bond between theirrespective GDF15 regions.

More particularly, in a specific embodiment, the tetramer comprises:

(a) two Dh2CpmFc(+)(N297G)(S354C) domains (one each heterodimer)comprising the sequence of SEQ ID NO:296,

(b) two Dh2CpmFc(−)(N297G)(Y349C) domains (one each heterodimer)comprising the sequence of SEQ ID NO:183, and

(c) two GDF15(N3D) polypeptide (one each heterodimer) comprising thesequence of SEQ ID NO:52.

In a preferred embodiment, the first polypeptide chain comprises theamino acid sequence:

(SEQ ID NO: 188) PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGARDGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 187) ccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacgggagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtgcaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtgcgcgcgacggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaaga ctgccactgcata.

In a preferred embodiment, the second polypeptide chain comprises theamino acid sequence of SEQ ID NO:182, which is encoded by the nucleicacid sequence of SEQ ID NO:186.

As discussed above, a tetramer is provided comprising two polypeptidechains comprising the sequence of SEQ ID NO:188 and two polypeptidechains comprising the sequence of SEQ ID NO:182.

II.H.22 Dh2CpmFc(−)(N297G)(A287C)-GDF15(Ndel3):Dh2CpmFc(+)(N297G)(L306C)

The designation“Dh2CpmFc(−)(N297G)(A287C)-GDF15(Ndel3):Dh2CpmFc(+)(N297G)(L306C)” inthe instant disclosure refers to a heterodimer comprising (i) a firstpolypeptide chain comprising GDF15(Ndel3) polypeptide, the N-terminus ofwhich is linked directly to the C-terminus of aDh2CpmFc(−)(N297G)(A287C) domain, and (ii) a second polypeptide chaincomprising a Dh2CpmFc(+)(N297G)(L306C) domain. The cysteine clampmutations allow the first and second polypeptide chains to be linked viaan interchain disulfide bond between C287 of the first monomer and C306of the second monomer.

In certain embodiments, a tetramer is provided, comprising a dimer oftwo Dh2CpmFc(−)(N297G)(A287C)-GDF15(Ndel3):Dh2CpmFc(+)(N297G)(L306C)heterodimers in which the two first polypeptide chains of eachheterodimer are linked via an interchain disulfide bond between theirrespective GDF15 regions.

More particularly, in a specific embodiment, a tetramer is providedwhich comprises:

(a) two Dh2CpmFc(+)(N297G)(L306C) domains (one each heterodimer)comprising the sequence:

(SEQ ID NO: 297) PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNCKTKPREEQYGSTYRVVSVCTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG,

(b) two Dh2CpmFc(−)(N297G)(A287C) (one each heterodimer) comprising thesequence:

(SEQ ID NO: 192) PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNCKTKPREEQYGSTYRVVSVCTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG,and

(c) two GDF15(Ndel3) polypeptides (one each heterodimer) comprising thesequence of SEQ ID NO:55.

In a preferred embodiment, the first polypeptide chain comprises theamino acid sequence:

(SEQ ID NO: 194) PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNCKTKPREEQYGSTYRVVSVCTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDT GVSLQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 193) ccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataattgcaagacaaagccgcgggaggagcagtacggcagcacgtaccgtgtggtcagcgtctgcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagact gccactgcata.

In a preferred embodiment, the second polypeptide chain comprises theamino acid sequence:

(SEQ ID NO: 191) PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNCKTKPREEQYGSTYRVVSVCTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 195) ccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataattgcaagacaaagccgcgggaggagcagtacggcagcacgtaccgtgtggtcagcgtctgcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccggaaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctgaagtccgacggctccttcttcctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa.

As discussed above, a tetramer is provided comprising two polypeptidechains comprising the sequence of SEQ ID NO:194 and two polypeptidechains comprising the sequence of SEQ ID NO:191.

II.H.23 Dh2CpmFc(−)(N297G)(A287C)-GDF15(N3D):Dh2CpmFc(+)(N297G)(L306C)

The designation“Dh2CpmFc(−)(N297G)(A287C)-GDF15(N3D):Dh2CpmFc(+)(N297G)(L306C)” in theinstant disclosure refers to a heterodimer comprising (i) a firstpolypeptide chain comprising a GDF15(N3D) polypeptide, the N-terminus ofwhich is linked directly to the C-terminus of aDh2CpmFc(−)(N297G)(A287C) domain, and (ii) a second polypeptide chaincomprising a Dh2CpmFc(+)(N297G)(L306C) domain. The cysteine clampmutations allow the first and second polypeptide chains to be linked viaan interchain disulfide bond between C287 of the first polypeptide chainand C306 of the second polypeptide chain.

In certain embodiments, a tetramer is provided, comprising a dimer oftwo Dh2CpmFc(−)(N297G)(A287C)-GDF15(N3D):Dh2CpmFc(+)(N297G)(L306C)heterodimers in which the two first polypeptide chains of eachheterodimer are linked via an interchain disulfide bond between theirrespective GDF15 regions.

More particularly, in a specific embodiment, a tetramer is providedwhich comprises:

(a) two Dh2CpmFc(+)(N297G)(L306C) domains (one each heterodimer)comprising the sequence of SEQ ID NO:297,

(b) two Dh2CpmFc(−)(N297G)(A287C) domains (one each heterodimer)comprising the sequence of SEQ ID NO:192, and

(c) two GDF15(N3D) polypeptides (one each heterodimer) comprising thesequence of SEQ ID NO:52.

In a preferred embodiment, the first polypeptide chain comprises theamino acid sequence:

(SEQ ID NO: 197) PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNCKTKPREEQYGSTYRVVSVCTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGARDGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 196) ccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataattgcaagacaaagccgcgggaggagcagtacggcagcacgtaccgtgtggtcagcgtctgcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtgcgcgcgacggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttag ccaaagactgccactgcata.

In a preferred embodiment, the second polypeptide chain comprises theamino acid sequence of SEQ ID NO:191, which is encoded by the nucleicacid sequence of SEQ ID NO:195.

As discussed above, a tetramer is provided comprising two polypeptidechains comprising the sequence of SEQ ID NO:197 and two polypeptidechains comprising the sequence of SEQ ID NO:191.

II.H.24Dh2CpmFc(−)(N297G)(A287C)(Y349C)-GDF15(Ndel3):Dh2CpmFc(+)(N297G)(L306C)(S354C)

The designation“Dh2CpmFc(−)(N297G)(A287C)(Y349C)-GDF15(Ndel3):Dh2CpmFc(+)(N297G)(L306C)(S354C)” in the instant disclosure refers to a heterodimer comprising (i)a first polypeptide chain comprising GDF15(Ndel3) polypeptide, theN-terminus of which is linked directly to the C-terminus of aDh2CpmFc(−)(N297G)(A287C)(Y349C) domain, and (ii) a second polypeptidechain comprising a Dh2CpmFc(+)(N297G)(L306C)(S354C) domain. The cysteineclamp mutations allow the first and second polypeptide chains to belinked via an interchain disulfide bond between C287 of the firstpolypeptide chain and C306 of the second polypeptide chain and betweenthe C349 of the first polypeptide chain and C349 of the secondpolypeptide chain.

In certain embodiments, a tetramer is provided, comprising a dimer oftwoDh2CpmFc(−)(N297G)(A287C)(Y349C)-GDF15(Ndel3):Dh2CpmFc(+)(N297G)(L306C)(S354C)heterodimers in which the two first polypeptide chains of eachheterodimer are linked via an interchain disulfide bond between theirrespective GDF15 regions.

More particularly, in a specific embodiment, a tetramer is providedwhich comprises:

(a) two Dh2CpmFc(+)(N297G)(L306C)(S354C) domains (one each heterodimer)comprising the sequence:

(SEQ ID NO: 298) PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNCKTKPREEQYGSTYRVVSVCTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG,

(b) two Dh2CpmFc(−)(N297G)(A287C)(Y349C) domains (one each heterodimer)comprising the sequence:

(SEQ ID NO: 199) PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNCKTKPREEQYGSTYRVVSVCTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG,and

(c) two GDF15(Ndel3) polypeptides (one each heterodimer) comprising thesequence of SEQ ID NO:55.

In a preferred embodiment, the first polypeptide chain comprises theamino acid sequence:

(SEQ ID NO: 201) PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNCKTKPREEQYGSTYRVVSVCTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDT GVSLQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 200) ccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataattgcaagacaaagccgcgggaggagcagtacggcagcacgtaccgtgtggtcagcgtctgcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtgcaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagact gccactgcata.

In a preferred embodiment, the second polypeptide chain comprises theamino acid sequence:

(SEQ ID NO: 198) PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNCKTKPREEQYGSTYRVVSVCTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 202) ccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataattgcaagacaaagccgcgggaggagcagtacggcagcacgtaccgtgtggtcagcgtctgcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatgccggaaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctgaagtccgacggctccttcttcctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa.

As discussed above, a tetramer is provided comprising two polypeptidechains comprising the sequence of SEQ ID NO:201 and two polypeptidechains comprising the sequence of SEQ ID NO:198.

II.H.25Dh2CpmFc(−)(N297G)(A287C)(Y349C)-GDF15(N3D):Dh2CpmFc(+)(N297G)(L306C)(S354C)

The designation“Dh2CpmFc(−)(N297G)(A287C)(Y349C)-GDF15(N3D):Dh2CpmFc(+)(N297G)(L306C)(S354C)”in the instant disclosure refers to a heterodimer comprising (i) a firstmonomer comprising GDF15(N3D) polypeptide, the N-terminus of which islinked directly to the C-terminus of a Dh2CpmFc(−)(N297G)(A287C)(Y349C)domain, and (ii) a second polypeptide chain comprising aDh2CpmFc(+)(N297G)(L306C)(S354C) domain. The cysteine clamp mutationsallow the first and second polypeptide chains to be linked via aninterchain disulfide bond between C287 of the first polypeptide chainand C306 of the second polypeptide chain and between the C349 of thefirst polypeptide chain and C349 of the second polypeptide chain.

In certain embodiments, a tetramer is provided, comprising a dimer oftwoDh2CpmFc(−)(N297G)(A287C)(Y349C)-GDF15(N3D):Dh2CpmFc(+)(N297G)(L306C)(S354C)heterodimers in which the two first polypeptide chains of eachheterodimer are linked via an interchain disulfide bond between theirrespective GDF15 regions.

More particularly, in a specific embodiment, a tetramer is providedwhich comprises:

(a) two Dh2CpmFc(+)(N297G)(L306C)(S354C) domains (one each heterodimer)comprising the sequence of SEQ ID NO:298,

(b) two Dh2CpmFc(−)(N297G)(A287C)(Y349C) domains (one each heterodimer)comprising the sequence of SEQ ID NO:199, and

(c) two GDF15(N3D) polypeptides (one each heterodimer) comprising thesequence of (SEQ ID NO:52).

In a preferred embodiment, the first polypeptide chain comprises theamino acid sequence:

(SEQ ID NO: 204) PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNCKTKPREEQYGSTYRVVSVCTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGARDGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 203) ccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataattgcaagacaaagccgcgggaggagcagtacggcagcacgtaccgtgtggtcagcgtctgcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtgcaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtgcgcgcgacggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttag ccaaagactgccactgcata.

In a preferred embodiment, the second polypeptide chain comprises theamino acid sequence of SEQ ID NO:198, which is encoded by the nucleicacid sequence of SEQ ID NO:202.

As discussed above, a tetramer is provided comprising two polypeptidechains comprising the sequence of SEQ ID NO:204 and two polypeptidechains comprising the sequence of SEQ ID NO:198.

II.H.26 GG-Dh2CpmFc(−)-GDF15(Ndel3):GG-Dh2CpmFc(+)

The designation “GG-Dh2CpmFc(−)-GDF15(Ndel3):GG-Dh2CpmFc(+)” in theinstant disclosure refers to a heterodimer comprising (i) a firstpolypeptide chain comprising a GDF15(Ndel3) polypeptide, the N-terminusof which is linked directly to the C-terminus of a GG-Dh2CpmFc(−)domain, and (ii) a second polypeptide chain comprising a GG-Dh2CpmFc(+)domain.

In certain embodiments, a tetramer is provided, comprising a dimer oftwo GG-Dh2CpmFc(−)-GDF15(Ndel3):GG-Dh2CpmFc(+) heterodimers in which thetwo first polypeptide chains of each heterodimer are linked via aninterchain disulfide bond between their respective GDF15 regions.

More particularly, in a specific embodiment, the tetramer comprises:

(a) two GG-Dh2CpmFc(+) domains (one each heterodimer) comprising thesequence:

(SEQ ID NO: 299) GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG,

(b) two GG-Dh2CpmFc(−) domains (one each heterodimer) comprising thesequence:

(SEQ ID NO: 206) GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG,and

(c) two GDF15(Ndel3) polypeptides (one each heterodimer) comprising thesequence of SEQ ID NO:55.

In a preferred embodiment, the first polypeptide chain comprises theamino acid sequence:

(SEQ ID NO: 208) GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKT DTGVSLQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 207) ggtggcccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagcca aagactgccactgcata.

In an embodiment employing the VH21 signal sequence, in a preferredembodiment, the first polypeptide chain comprises the amino acidsequence (signal sequence single underlined):

(SEQ ID NO: 243) MEWSWVFLFFLSVTTGVHSGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence (signal sequenceunderlined):

(SEQ ID NO: 244) atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactccggtggcccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagcca aagactgccactgcata.

In a preferred embodiment, the second polypeptide chain comprises theamino acid sequence:

(SEQ ID NO: 205) GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 209) ggtggcccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccggaaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctgaagtccgacggctccttcttcctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa.

In an embodiment employing the VH21 signal sequence, in a preferredembodiment, the second polypeptide chain comprises the amino acidsequence (signal sequence single underlined):

(SEQ ID NO: 245) MEWSWVFLFFLSVTTGVHSGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK,which is encoded by the nucleic acid sequence (signal sequenceunderlined):

(SEQ ID NO: 246) atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactccggtggcccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccggaaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctgaagtccgacggctccttcttcctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa.

As discussed above, a tetramer is provided comprising two polypeptidechains comprising the sequence of SEQ ID NO:208 and two polypeptidechains comprising the sequence of SEQ ID NO:205.

II.H.27 GG-Dh2CpmFc(−)-GDF15(N3D):GG-Dh2CpmFc(+)

The designation “GG-Dh2CpmFc(−)-GDF15(N3D):GG-DhCpmFc(+)” in the instantdisclosure refers to a heterodimer comprising (i) a first polypeptidechain comprising a GDF15(N3D) polypeptide, the N-terminus of which islinked directly to the C-terminus of a GG-Dh2CpmFc(−) domain, and (ii) asecond polypeptide chain comprising a GG-DhCpmFc(+) domain.

In certain embodiments, a tetramer is provided, comprising a dimer oftwo GG-Dh2CpmFc(−)-GDF15(N3D):GG-Dh2CpmFc(+) heterodimers in which thetwo first polypeptide chains of each heterodimer are linked via aninterchain disulfide bond between their respective GDF15 regions.

More particularly, in a specific embodiment, the tetramer comprises:

(a) two GG-Dh2CpmFc(+) domains (one each heterodimer) comprising thesequence of SEQ ID NO:299,

(b) two GG-Dh2CpmFc(−) domains (one each heterodimer) comprising thesequence of SEQ ID NO:206, and

(c) two GDF15(N3D) polypeptides (one each heterodimer) comprising thesequence of SEQ ID NO:52.

In a preferred embodiment, the first polypeptide chain comprises theamino acid sequence:

(SEQ ID NO: 211) GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGARDGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 210) ggtggcccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtgcgcgcgacggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcata.

In an embodiment employing the VH21 signal sequence, in a preferredembodiment, the first polypeptide chain comprises the amino acidsequence (signal sequence single underlined):

(SEQ ID NO: 247) MEWSWVFLFFLSVTTGVHSGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGARDGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence (signal sequenceunderlined):

(SEQ ID NO: 248) atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactccggtggcccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtgcgcgcgacggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcata.

In a preferred embodiment, the second polypeptide chain comprises theamino acid sequence of SEQ ID NO:205, which is encoded by the nucleicacid sequence of SEQ ID NO:209.

In an embodiment employing the VH21 signal sequence, in a preferredembodiment, the second polypeptide chain comprises the amino acidsequence of SEQ ID NO:245, which is encoded by the nucleic acid sequenceof SEQ ID NO:246.

As discussed above, a tetramer is provided comprising two polypeptidechains comprising the sequence of SEQ ID NO:211 and two polypeptidechains comprising the sequence of SEQ ID NO:205.

II.H.28 GG-Dh2CpmFc(−)(Y349C)-GDF15(Ndel3):GG-Dh2CpmFc(+)(S354C)

The designation“GG-Dh2CpmFc(−)(Y349C)-GDF15(Ndel3):GG-Dh2CpmFc(+)(S354C)” in theinstant disclosure refers to a heterodimer, which comprises (i) a firstpolypeptide chain comprising GDF15(Ndel3) polypeptide, the N-terminus ofwhich is linked directly to the C-terminus of a GG-Dh2CpmFc(−)(Y349C)domain, and (ii) a second polypeptide chain comprising aGG-Dh2CpmFc(+)(S345C) domain. The cysteine clamp mutations allow thefirst and second polypeptide chains to be linked via an interchaindisulfide bond between C349 of the first polypeptide chain and C354 ofthe second polypeptide chain.

In certain embodiments, a tetramer is provided, comprising a dimer oftwo GG-Dh2CpmFc(−)(Y349C)-GDF15(Ndel3):GG-Dh2CpmFc(+)(S354C)heterodimers in which the two first polypeptide chains of eachheterodimer are linked via an interchain disulfide bond between theirrespective GDF15 regions.

More particularly, in a specific embodiment, the tetramer comprises:

(a) two GG-Dh2CpmFc(+)(S354C) (one each heterodimer) comprising thesequence:

(SEQ ID NO: 300) GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG,

(b) two GG-Dh2CpmFc(−)(Y349C) chains (one each heterodimer) comprisingthe sequence:

(SEQ ID NO: 213) GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG,and

(c) two GDF15(Ndel3) polypeptides (one each heterodimer) comprising thesequence of SEQ ID NO:55.

In a preferred embodiment, the first polypeptide chain comprises theamino acid sequence:

(SEQ ID NO: 215) GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKT DTGVSLQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 214) ggtggcccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtgcaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagcca aagactgccactgcata.

In an embodiment employing the VH21 signal sequence, in a preferredembodiment, the first polypeptide chain comprises the amino acidsequence (signal sequence single underlined):

(SEQ ID NO: 249) MEWSWVFLFFLSVTTGVHSGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence (signal sequenceunderlined):

(SEQ ID NO: 250) atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactccggtggcccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtgcaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagcca aagactgccactgcata.

In a preferred embodiment, the second polypeptide chain comprises theamino acid sequence:

(SEQ ID NO: 212) GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 216) ggtggcccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatgccggaaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctgaagtccgacggctccttcttcctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa.

In an embodiment employing the VH21 signal sequence, in a preferredembodiment, the second polypeptide chain comprises the amino acidsequence (signal sequence single underlined):

(SEQ ID NO: 251) MEWSWVFLFFLSVTTGVHSGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK,which is encoded by the nucleic acid sequence (signal sequenceunderlined):

(SEQ ID NO: 252) atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactccggtggcccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatgccggaaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctgaagtccgacggctccttcttcctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa.

As discussed above, a tetramer is provided comprising two polypeptidechains comprising the sequence of SEQ ID NO:215 and two polypeptidechains comprising the sequence of SEQ ID NO:212.

II.H.29 GG-Dh2CpmFc(−)(Y349C)-GDF15(N3D):GG-Dh2CpmFc(+)(S354C)

The designation “GG-Dh2CpmFc(−)(Y349C)-GDF15(N3D):GG-Dh2CpmFc(+)(S354C)”in the instant disclosure refers to a heterodimer, which comprises (i) afirst polypeptide chain comprising a GDF15(N3D) polypeptide, theN-terminus of which is linked directly to the C-terminus of aGG-Dh2CpmFc(−)(Y349C) domain, and (ii) a second polypeptide chaincomprising a GG-Dh2CpmFc(+)(S345C) domain. The cysteine clamp mutationsallow the first and second polypeptide chains to be linked via aninterchain disulfide bond between C349 of the first polypeptide chainand C354 of the second polypeptide chain.

In certain embodiments, a tetramer is provided, comprising a dimer oftwo GG-Dh2CpmFc(−)(Y349C)-GDF15(N3D):GG-Dh2CpmFc(+)(S354C) heterodimersin which the two first polypeptide chains of each heterodimer are linkedvia an interchain disulfide bond between their respective GDF15 regions.

More particularly, in a specific embodiment, the tetramer comprises:

(a) two GG-Dh2CpmFc(+)(S354C) domains (one each heterodimer) comprisingthe sequence of SEQ ID NO:300,

(b) two GG-Dh2CpmFc(−)(Y349C) domains (one each heterodimer) comprisingthe sequence of SEQ ID NO:213, and

(c) two GDF15(N3D) polypeptides (one each heterodimer) comprising thesequence of SEQ ID NO:52.

In a preferred embodiment, the first polypeptide chain comprises theamino acid sequence:

(SEQ ID NO: 218) GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGARDGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 217) ggtggcccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtgcaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtgcgcgcgacggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcata.

In an embodiment employing the VH21 signal sequence, in a preferredembodiment, the first polypeptide chain comprises the amino acidsequence (signal sequence single underlined):

(SEQ ID NO: 253) MEWSWVFLFFLSVTTGVHSGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGARDGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence (signal sequenceunderlined):

(SEQ ID NO: 254) atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactccggtggcccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtgcaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtgcgcgcgacggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcata.

In a preferred embodiment, the second polypeptide chain comprises theamino acid sequence of SEQ ID NO:212, which is encoded by the nucleicacid sequence of SEQ ID NO:218.

In an embodiment employing the VH21 signal sequence, in a preferredembodiment, the second polypeptide chain comprises the amino acidsequence of SEQ ID NO:251, which is encoded by the nucleic acid sequenceof SEQ ID NO:252.

As discussed above, a tetramer is provided comprising two polypeptidechains comprising the sequence of SEQ ID NO:218 and two polypeptidechains comprising the sequence of SEQ ID NO:212.

II.H.30 Dh3CpmFc(−)-GDF15(Ndel3):Dh3CpmFc(+)

The designation “Dh3CpmFc(−)-GDF15(Ndel3):Dh3CpmFc(+)” in the instantdisclosure refers to a heterodimer, which comprises (i) a firstpolypeptide chain comprising a GDF15(Ndel3) polypeptide, the N-terminusof which is linked directly to the C-terminus vof a Dh3CpmFc(−) domain,and (ii) a second polypeptide chain comprising a Dh3CpmFc(+) domain.

In certain embodiments, a tetramer is provided, comprising a dimer oftwo Dh3CpmFc(−)-GDF15(Ndel3):Dh3CpmFc(+) heterodimers in which the twofirst polypeptide chains of each heterodimer are linked via aninterchain disulfide bond between their respective GDF15 regions.

More particularly, in a specific embodiment, the tetramer comprises:

(a) two Dh3CpmFc(+) domains (one each heterodimer) comprising thesequence:

(SEQ ID NO: 301) GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPG,

(b) two Dh3CpmFc(−) domains (one each heterodimer) comprising thesequence:

(SEQ ID NO: 220) GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPG,and

(c) two GDF15(Ndel3) polypeptides (one each heterodimer) comprising thesequence of SEQ ID NO:55.

In a preferred embodiment, the first polypeptide chain comprises theamino acid sequence:

(SEQ ID NO: 222) GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTD TGVSLQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 221) ggcccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcata.

In an embodiment employing the VK1 signal sequence, in a preferredembodiment, the first polypeptide chain comprises the amino acidsequence (signal sequence single underlined):

(SEQ ID NO: 255) MDMRVPAQLLGLLLLWLRGARCGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence (signal sequenceunderlined):

(SEQ ID NO: 256) atggacatgagggtgcccgctcagctcctggggctcctgctgctgtggctgagaggtgcgcgctgtggcccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcata.

In a preferred embodiment, the second polypeptide chain comprises theamino acid sequence:

(SEQ ID NO: 219) GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 223) ggcccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccggaaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctgaagtccgacggctccttcttcctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa.

In an embodiment employing the VK1 signal sequence, in a preferredembodiment, the second polypeptide chain comprises the amino acidsequence (signal sequence single underlined):

(SEQ ID NO: 257) MDMRVPAQLLGLLLLWLRGARCGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK,which is encoded by the nucleic acid sequence (signal sequenceunderlined):

(SEQ ID NO: 258) atggacatgagggtgcccgctcagctcctggggctcctgctgctgtggctgagaggtgcgcgctgtggcccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccggaaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctgaagtccgacggctccttcttcctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa.

As discussed above, a tetramer is provided comprising two polypeptidechains comprising the sequence of SEQ ID NO:222 and two polypeptidechains comprising the sequence of SEQ ID NO:219.

II.H.31 Dh3CpmFc(−)-GDF15(N3D):Dh3CpmFc(+)

The designation “Dh3CpmFc(−)-GDF15(N3D):Dh3CpmFc(+)” in the instantdisclosure refers to a heterodimer comprising (i) a first polypeptidechain comprising a GDF15(N3D) polypeptide, the N-terminus of which islinked directly to the C-terminus of a Dh3CpmFc(−) domain, and (ii) asecond polypeptide chain comprising a Dh3CpmFc(+) domain.

In certain embodiments, a tetramer is provided, comprising a dimer oftwo Dh3CpmFc(−)-GDF15(N3D):Dh3CpmFc(+) heterodimers in which the twofirst polypeptide chains of each heterodimer are linked via aninterchain disulfide bond between their respective GDF15 regions.

More particularly, in a specific embodiment, the tetramer comprises:

(a) two Dh3CpmFc(+) domains (one each heterodimer) comprising thesequence of SEQ ID NO:301,

(b) two Dh3CpmFc(−) (one each heterodimer) comprising the sequence ofSEQ ID NO:220, and

(c) two GDF15(N3D) polypeptides (one each heterodimer) comprising thesequence of SEQ ID NO:52.

In a preferred embodiment, the first polypeptide chain comprises theamino acid sequence:

(SEQ ID NO: 225) GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGARDGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 224) atggacatgagggtgcccgctcagctcctggggctcctgctgctgtggctgagaggtgcgcgctgtggcccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtgcgcgcgacggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcata.

In an embodiment employing the VK1 signal sequence, in a preferredembodiment, the first polypeptide chain comprises the amino acidsequence (signal sequence single underlined):

(SEQ ID NO: 259) MDMRVPAQLLGLLLLWLRGARCGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGARDGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence (signal sequenceunderlined):

(SEQ ID NO: 260) atggacatgagggtgcccgctcagctcctggggctcctgctgctgtggctgagaggtgcgcgctgtggcccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtgcgcgcgacggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcata.

In a preferred embodiment, the second polypeptide chain comprises theamino acid sequence of SEQ ID NO:219, which is encoded by the nucleicacid sequence of SEQ ID NO:223.

In an embodiment employing the VK1 signal sequence, in a preferredembodiment, the second polypeptide chain comprises the amino acidsequence of SEQ ID NO:257, which is encoded by the nucleic acid sequenceof SEQ ID NO:258.

As discussed above, a tetramer is provided comprising two polypeptidechains comprising the sequence of SEQ ID NO:225 and two polypeptidechains comprising the sequence of SEQ ID NO:219.

II.H.32 Dh3CpmFc(−)(Y349C)-GDF15(Ndel3):Dh3CpmFc(+)(S354C)

The designation “Dh3CpmFc(−)(Y349C)-GDF15(Ndel3):Dh3CpmFc(+)(S354C)” inthe instant disclosure refers to a heterodimer, which comprises (i) afirst polypeptide chain comprising a GDF15(Ndel3) polypeptide, theN-terminus of which is linked directly to the C-terminus of aDh3CpmFc(−)(Y349C) domain, and (ii) a second polypeptide chaincomprising a Dh3CpmFc(+)(S345C) domain. The cysteine clamp mutationsallow the first and second polypeptide chains to be linked via aninterchain disulfide bond between C349 of the first polypeptide and C354of the second polypeptide.

In certain embodiments, a tetramer is provided, comprising a dimer oftwo Dh3CpmFc(−)(Y349C)-GDF15(Ndel3):Dh3CpmFc(+)(S354C) heterodimers inwhich the two first polypeptide chains of each heterodimer are linkedvia an interchain disulfide bond between their respective GDF15 regions.

More particularly, in a specific embodiment, the tetramer comprises:

(a) two Dh3CpmFc(+)(S354C) domains (one each heterodimer) comprising thesequence:

(SEQ ID NO: 302) GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPG,

(b) two Dh3CpmFc(−)(Y349C) domains (one each heterodimer) comprising thesequence:

(SEQ ID NO: 227) GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPG,and

(c) two GDF15(Ndel3) polypeptides (one each heterodimer) comprising thesequence of SEQ ID NO:55.

In a preferred embodiment, the first polypeptide chain comprises theamino acid sequence:

(SEQ ID NO: 229) GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTD TGVSLQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 228) atggacatgagggtgcccgctcagctcctggggctcctgctgctgtggctgagaggtgcgcgctgtggcccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtgcaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcata.

In an embodiment employing the VK1 signal sequence, in a preferredembodiment, the first polypeptide chain comprises the amino acidsequence (signal sequence single underlined):

(SEQ ID NO: 261) MDMRVPAQLLGLLLLWLRGARCGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence (signal sequenceunderlined):

(SEQ ID NO: 262) atggacatgagggtgcccgctcagctcctggggctcctgctgctgtggctgagaggtgcgcgctgtggcccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtgcaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcata.

In a preferred embodiment, the second polypeptide chain comprises theamino acid sequence:

(SEQ ID NO: 226) GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 230) ggcccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatgccggaaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctgaagtccgacggctccttcttcctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa.

In an embodiment employing the VK1 signal sequence, in a preferredembodiment, the second polypeptide chain comprises the amino acidsequence (signal sequence single underlined):

(SEQ ID NO: 263) MDMRVPAQLLGLLLLWLRGARCGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK,which is encoded by the nucleic acid sequence (signal sequenceunderlined):

(SEQ ID NO: 264) atggacatgagggtgcccgctcagctcctggggctcctgctgctgtggctgagaggtgcgcgctgtggcccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatgccggaaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctgaagtccgacggctccttcttcctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa.

As discussed above, a tetramer is provided comprising two polypeptidechains comprising the sequence of SEQ ID NO:229 and two polypeptidechains comprising the sequence of SEQ ID NO:226.

II.H.33 Dh3CpmFc(−)(Y349C)-GDF15(N3D):Dh3CpmFc(+)(S354C)

The designation “Dh3CpmFc(−)(Y349C)-GDF15(N3D):Dh3CpmFc(+)(S354C)” inthe instant disclosure refers to a heterodimer, which comprises (i) afirst polypeptide chain comprising a GDF15(N3D) polypeptide, theN-terminus of which is linked directly to the C-terminus of aDh3CpmFc(−)(Y349C) domain, and (ii) a second polypeptide chaincomprising a Dh3CpmFc(+)(S345C) domain. The cysteine clamp mutationsallow the first and second polypeptide chains to be linked via aninterchain disulfide bond between C349 of the first polypeptide chainand C354 of the second polypeptide chain.

In certain embodiments, a tetramer is provided, comprising a dimer oftwo Dh3CpmFc(−)(Y349C)-GDF15(N3D):Dh3CpmFc(+)(S354C) heterodimers inwhich the two first polypeptide chains of each heterodimer are linkedvia an interchain disulfide bond between their respective GDF15 regions.

More particularly, in a specific embodiment, the tetramer comprises:

(a) two Dh3CpmFc(+)(S354C) domains (one each heterodimer) comprising thesequence of SEQ ID NO:302,

(b) two Dh3CpmFc(−)(Y349C) domains (one each heterodimer) comprising thesequence of SEQ ID NO:227, and

(c) two GDF15(N3D) polypeptides (one each heterodimer) comprising thesequence of SEQ ID NO:52.

In a preferred embodiment, the first polypeptide chain comprises theamino acid sequence:

(SEQ ID NO: 232) GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGARDGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 231) ggcccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtgcaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtgcgcgcgacggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcata.

In an embodiment employing the VK1 signal sequence, in a preferredembodiment, the first polypeptide chain comprises the amino acidsequence (signal sequence single underlined):

(SEQ ID NO: 265) MDMRVPAQLLGLLLLWLRGARCGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGARDGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence (signal sequenceunderlined):

(SEQ ID NO: 266) atggacatgagggtgcccgctcagctcctggggctcctgctgctgtggctgagaggtgcgcgctgtggcccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtgcaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtgcgcgcgacggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcata.

In a preferred embodiment, the second polypeptide chain comprises theamino acid sequence of SEQ ID NO:226, which is encoded by the nucleicacid sequence of SEQ ID NO:230.

In an embodiment employing the VK1 signal sequence, in a preferredembodiment, the second polypeptide chain comprises the amino acidsequence of SEQ ID NO:263, which is encoded by the nucleic acid sequenceof SEQ ID NO:264.

As discussed above, a tetramer is provided comprising two polypeptidechains comprising the sequence of SEQ ID NO:232 and two polypeptidechains comprising the sequence of SEQ ID NO:226.

II.H.34 DhMonoFc(N297G)-GDF15

The designation “DhMonoFc(N297G)-GDF15” in the instant disclosure refersto a fusion protein comprising a GDF15 polypeptide, the N-terminus ofwhich is linked directly to the C-terminus of a DhMonoFc(N297G) domainby a peptide bond.

In certain embodiments, a homodimer is provided comprising two suchfusion proteins linked via interchain disulfide bond between theirrespective GDF15 polypeptides.

More particularly, in a specific embodiment, the homodimer comprises:

(a) two DhMonoFc(N297G) domains (one each monomer) comprising thesequence:

(SEQ ID NO: 233) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVTTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPG;and

(b) two GDF15 polypeptides (one each heterodimer) comprising thesequence of SEQ ID NO:12.

In a preferred embodiment, the fusion protein comprises the amino acidsequence:

(SEQ ID NO: 235) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVTTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 234) gcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacggcagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgaccaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtgcgcgcaacggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcata.

As discussed above, in a specific embodiment, a homodimer is providedcomprising two monomers having the sequence of SEQ ID NO:235.

II.H.35 DhMonoFc(N297G)-(G₄S)₄-GDF15

The designation “DhMonoFc(N297G)-(G₄S)₄-GDF15” in the instant disclosurerefers to a fusion protein comprising a GDF15 polypeptide linked to aDhMonoFc(N297G) domain via a linker comprising the sequence of SEQ IDNO:18 that connects the N-terminus of the GDF15 polypeptide to theC-terminus of the DhMonoFc(N297G) domain by a peptide bond.

In certain embodiments, a homodimer is provided comprising two suchfusion proteins linked via interchain disulfide bond between theirrespective GDF15 polypeptides.

More particularly, in a specific embodiment, the homodimer comprises:

(a) two DhMonoFc(N297G) domains (one each monomer) comprising thesequence:

(SEQ ID NO: 236) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVTTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPG;

(b) two GDF15 polypeptides (one each monomer) comprising the sequence ofSEQ ID NO:12; and

(c) two polypeptide linkers (one each monomer) comprising the sequenceof SEQ ID NO:18 each linking the N-terminus of a GDF15 polypeptide tothe C-terminus of a DhMonoFc(N297G) domain via peptide bonds.

In a preferred embodiment, the fusion protein comprises the amino acidsequence (linker double underlined):

(SEQ ID NO: 238) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVTTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 237) gcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacggcagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgaccaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtggaggtggtggatccggaggcggtggaagcggaggtggtggatctggaggcggtggaagcgcgcgcaacggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcat a.

As discussed above, in a specific embodiment, a homodimer is providedcomprising two monomers having the sequence of SEQ ID NO:238.

II.H.36 DhMonoFc(N297G)-G₄-GDF15

The designation “DhMonoFc(N297G)-G₄-GDF15” in the instant disclosurerefers to a fusion protein comprising a GDF15 polypeptide linked to aDhMonoFc(N297G) domain via a linker comprising the sequence of SEQ IDNO:58 that connects the N-terminus of the GDF15 polypeptide to theC-terminus of the DhMonoFc(N297G) domain by a peptide bond.

In certain embodiments, a homodimer is provided comprising two suchfusion proteins linked via interchain disulfide bond between theirrespective GDF15 polypeptides.

More particularly, in a specific embodiment, the homodimer comprises:

(a) two DhMonoFc(N297G) domains (one each monomer) comprising thesequence of SEQ ID NO:236;

(b) two GDF15 polypeptides (one each monomer) comprising the sequence ofSEQ ID NO:12; and

(c) two polypeptide linkers (one each monomer) comprising the sequenceof SEQ ID NO:58 each linking the N-terminus of a GDF15 polypeptide tothe C-terminus of a DhMonoFc(N297G) domain by peptide bonds.

In a preferred embodiment, the fusion protein comprises the amino acidsequence (linker double underlined):

(SEQ ID NO: 240) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVTTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI,which is encoded by the nucleic acid sequence:

(SEQ ID NO: 239) gcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacggcagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgaccaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgacaccacgcctcccgtgctggactccgacggctccttcttcctctatagcgacctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtggaggtggtggagcgcgcaacggagaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgc ata.

As discussed above, in a specific embodiment, a homodimer is providedcomprising two monomers having the sequence of SEQ ID NO:240.

III. GDF15 Polypeptides and Constructs Comprising GDF15, IncludingMutant Forms Thereof

As disclosed herein, the GDF15 polypeptides (including the full lengthand mature forms of human GDF15) and the constructs comprising GDF15described in the instant disclosure can be engineered and/or producedusing standard molecular biology methodology to form a mutant form ofthe GDF15 polypeptides and constructs provided herein. In variousexamples, a nucleic acid sequence encoding a mutant form of the GDF15polypeptides and constructs provided herein, which can comprise all or aportion of SEQ ID NOs:4, 8 or 12 can be isolated and/or amplified fromgenomic DNA, or cDNA using appropriate oligonucleotide primers. Primerscan be designed based on the nucleic and amino acid sequences providedherein according to standard (RT)-PCR amplification techniques. Theamplified GDF15 mutant polypeptide nucleic acid can then be cloned intoa suitable vector and characterized by DNA sequence analysis.

Oligonucleotides for use as probes in isolating or amplifying all or aportion of a mutant form of the GDF15 polypeptides and constructsprovided herein can be designed and generated using standard synthetictechniques, e.g., automated DNA synthesis apparatus, or can be isolatedfrom a longer sequence of DNA.

III.A. GDF15 Polypeptide and Polynucleotide Sequences

In vivo, GDF15 is expressed as a contiguous amino acid sequencecomprising a signal sequence, a pro domain and an active domain.

The 308 amino acid sequence of full length human GDF15 is:

(SEQ ID NO: 4) MPGQELRTVNGSQMLLVLLVLSWLPHGGALSLAEASRASFPGPSELHSEDSRFRELRKRYEDLLTRLRANQSWEDSNTDLVPAPAVRILTPEVRLGSGGHLHLRISRAALPEGLPEASRLHRALFRLSPTASRSWDVTRPLRRQLSLARPQAPALHLRLSPPPSQSDQLLAESSSARPQLELHLRPQAARGRRRARARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDL LAKDCHCIand is encoded by the DNA sequence:

(SEQ ID NO: 3) atgcccgggcaagaactcaggacggtgaatggctctcagatgctcctggtgttgctggtgctctcgtggctgccgcatgggggcgccctgtctctggccgaggcgagccgcgcaagtttcccgggaccctcagagttgcactccgaagactccagattccgagagttgcggaaacgctacgaggacctgctaaccaggctgcgggccaaccagagctgggaagattcgaacaccgacctcgtcccggcccctgcagtccggatactcacgccagaagtgcggctgggatccggcggccacctgcacctgcgtatctctcgggccgcccttcccgaggggctccccgaggcctcccgccttcaccgggctctgttccggctgtccccgacggcgtcaaggtcgtgggacgtgacacgaccgctgcggcgtcagctcagccttgcaagaccccaggcgcccgcgctgcacctgcgactgtcgccgccgccgtcgcagtcggaccaactgctggcagaatcttcgtccgcacggccccagctggagttgcacttgcggccgcaagccgccagggggcgccgcagagcgcgtgcgcgcaacggggaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcatatga.The 303 amino acid sequence of full length murine GDF15 is:

(SEQ ID NO: 6) MAPPALQAQPPGGSQLRFLLFLLLLLLLLSWPSQGDALAMPEQRPSGPESQLNADELRGRFQDLLSRLHANQSREDSNSEPSPDPAVRILSPEVRLGSHGQLLLRVNRASLSQGLPEAYRVHRALLLLTPTARPWDITRPLKRALSLRGPRAPALRLRLTPPPDLAMLPSGGTQLELRLRVAAGRGRRSAHAHPRDSCPLGPGRCCHLETVQATLEDLGWSDWVLSPRQLQLSMCVGECPHLYRSANTHAQIKARLHGLQPDKVPAPCCVPSSYTPVVLMHRTDSGVSLQTYDDLVARGC HCAand is encoded by the DNA sequence:

(SEQ ID NO: 5) atggccccgcccgcgctccaggcccagcctccaggcggctctcaactgaggttcctgctgttcctgctgctgttgctgctgctgctgtcatggccatcgcagggggacgccctggcaatgcctgaacagcgaccctccggccctgagtcccaactcaacgccgacgagctacggggtcgcttccaggacctgctgagccggctgcatgccaaccagagccgagaggactcgaactcagaaccaagtcctgacccagctgtccggatactcagtccagaggtgagattggggtcccacggccagctgctactccgcgtcaaccgggcgtcgctgagtcagggtctccccgaagcctaccgcgtgcaccgagcgctgctcctgctgacgccgacggcccgcccctgggacatcactaggcccctgaagcgtgcgctcagcctccggggaccccgtgctcccgcattacgcctgcgcctgacgccgcctccggacctggctatgctgccctctggcggcacgcagctggaactgcgcttacgggtagccgccggcagggggcgccgaagcgcgcatgcgcacccaagagactcgtgcccactgggtccggggcgctgctgtcacttggagactgtgcaggcaactcttgaagacttgggctggagcgactgggtgctgtccccgcgccagctgcagctgagcatgtgcgtgggcgagtgtccccacctgtatcgctccgcgaacacgcatgcgcagatcaaagcacgcctgcatggcctgcagcctgacaaggtgcctgccccgtgctgtgtcccctccagctacaccccggtggttcttatgcacaggacagacagtggtgtgtcactgcagacttatgatgacctggtggcccggggctgc cactgcgcttga.

The amino acid sequence of human GDF15 following cleavage of the 29residue signal sequence is:

(SEQ ID NO: 8) LSLAEASRASFPGPSELHSEDSRFRELRKRYEDLLTRLRANQSWEDSNTDLVPAPAVRILTPEVRLGSGGHLHLRISRAALPEGLPEASRLHRALFRLSPTASRSWDVTRPLRRQLSLARPQAPALHLRLSPPPSQSDQLLAESSSARPQLELHLRPQAARGRRRARARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCIand is encoded by the DNA sequence:

(SEQ ID NO: 7) ctgtctctggccgaggcgagccgcgcaagtttcccgggaccctcagagttgcactccgaagactccagattccgagagttgcggaaacgctacgaggacctgctaaccaggctgcgggccaaccagagctgggaagattcgaacaccgacctcgtcccggcccctgcagtccggatactcacgccagaagtgcggctgggatccggcggccacctgcacctgcgtatctctcgggccgcccttcccgaggggctccccgaggcctcccgccttcaccgggctctgttccggctgtccccgacggcgtcaaggtcgtgggacgtgacacgaccgctgcggcgtcagctcagccttgcaagaccccaggcgcccgcgctgcacctgcgactgtcgccgccgccgtcgcagtcggaccaactgctggcagaatcttcgtccgcacggccccagctggagttgcacttgcggccgcaagccgccagggggcgccgcagagcgcgtgcgcgcaacggggaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcatatga

The amino acid sequence of murine GDF15 following cleavage of the 32residue signal sequence is:

(SEQ ID NO: 10) SQGDALAMPEQRPSGPESQLNADELRGRFQDLLSRLHANQSREDSNSEPSPDPAVRILSPEVRLGSHGQLLLRVNRASLSQGLPEAYRVHRALLLLTPTARPWDITRPLKRALSLRGPRAPALRLRLTPPPDLAMLPSGGTQLELRLRVAAGRGRRSAHAHPRDSCPLGPGRCCHLETVQATLEDLGWSDWVLSPRQLQLSMCVGECPHLYRSANTHAQIKARLHGLQPDKVPAPCCVPSSYTPVVLMHR TDSGVSLQTYDDLVARGCHCAand is encoded by the DNA sequence:

(SEQ ID NO: 9) tcgcagggggacgccctggcaatgcctgaacagcgaccctccggccctgagtcccaactcaacgccgacgagctacggggtcgcttccaggacctgctgagccggctgcatgccaaccagagccgagaggactcgaactcagaaccaagtcctgacccagctgtccggatactcagtccagaggtgagattggggtcccacggccagctgctactccgcgtcaaccgggcgtcgctgagtcagggtctccccgaagcctaccgcgtgcaccgagcgctgctcctgctgacgccgacggcccgcccctgggacatcactaggcccctgaagcgtgcgctcagcctccggggaccccgtgctcccgcattacgcctgcgcctgacgccgcctccggacctggctatgctgccctctggcggcacgcagctggaactgcgcttacgggtagccgccggcagggggcgccgaagcgcgcatgcgcacccaagagactcgtgcccactgggtccggggcgctgctgtcacttggagactgtgcaggcaactcttgaagacttgggctggagcgactgggtgctgtccccgcgccagctgcagctgagcatgtgcgtgggcgagtgtccccacctgtatcgctccgcgaacacgcatgcgcagatcaaagcacgcctgcatggcctgcagcctgacaaggtgcctgccccgtgctgtgtcccctccagctacaccccggtggttcttatgcacaggacagacagtggtgtgtcactgcagacttatgatgacctggtggcccgggg ctgccactgcgcttga

The biologically active form of GDF15 comprises a homodimer comprisingtwo mature GDF15 monomers, each of which comprises SEQ ID NO:12. Themonomer that homodimerizes to form the native mature human GDF15 dimeris encoded by the nucleic acid sequence:

(SEQ ID NO: 11) gcgcgcaacggggaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcatatgaand comprises the amino acid sequence:

(SEQ ID NO: 12) ARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQT YDDLLAKDCHCI.

Thus, the “native mature human GDF15 dimer” comprises two covalentlyassociated monomers comprising SEQ ID NO:12.

The amino acid sequence of the recombinant active form of the humanGDF15, which comprises a homodimer comprising nine cysteines in eachmonomer to form one interchain disulfide bond and four intrachaindisulfide bonds (shown with an optional N-terminal methionine residue inparentheses), is:

(SEQ ID NO: 189) (M)ARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVS LQTYDDLLAKDCHCIand is encoded by the DNA sequence (shown with an optional N-terminalmethionine codon in parentheses):

(SEQ ID NO: 190) (atg)gcgcgcaacggggaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcatataa.

The amino acid sequence of the recombinant active form of the murineGDF15, which comprises a homodimer comprising nine cysteines in eachmonomer to form one interchain disulfide bond and four intrachaindisulfide bonds, is:

(SEQ ID NO: 14) (M)SAHAHPRDSCPLGPGRCCHLETVQATLEDLGWSDWVLSPRQLQLSMCVGECPHLYRSANTHAQIKARLHGLQPDKVPAPCCVPSSYTPVVLMHRTDS GVSLQTYDDLVARGCHCAand is encoded by the DNA sequence:

(SEQ ID NO: 13) (atg)agcgcgcatgcgcacccaagagactcgtgcccactgggtccggggcgctgctgtcacctggagactgtgcaggcaactcttgaagacttgggctggagcgactgggtgttgtccccgcgccagctgcagctgagcatgtgcgtgggcgagtgtccccacctgtatcgctccgcgaacacgcatgcgcagatcaaagcacgcctgcatggcctgcagcctgacaaggtgcctgccccgtgctgtgtcccctccagctacaccccggtggttcttatgcacaggacagacagtggtgtgtcactgcagacttatgatgacctggtggcccggggctgccactgcgct tga.

As stated herein, the term “GDF15 polypeptide” refers to a GDFpolypeptide comprising the human amino acid sequences SEQ ID NOs:4, 8and 12. The term “GDF15 mutant polypeptide,” however, encompassespolypeptides comprising an amino acid sequence that differs from theamino acid sequence of a naturally-occurring GDF polypeptide sequence,e.g., SEQ ID NOs: 4, 8 and 12, by one or more amino acids, such that thesequence is at least 85% identical to SEQ ID NOs: 4, 8 and 12. GDF15polypeptides can be generated by introducing one or more amino acidsubstitutions, either conservative or non-conservative and usingnaturally or non-naturally-occurring amino acids, at particularpositions of the GDF15 polypeptide, or by deleting particular residuesor stretches of residues.

A “conservative amino acid substitution” can involve a substitution of anative amino acid residue (i.e., a residue found in a given position ofthe wild-type GDF15 polypeptide sequence) with a non-native residue(i.e., a residue that is not found in a given position of the wild-typeGDF15 polypeptide sequence) such that there is little or no effect onthe polarity or charge of the amino acid residue at that position.Conservative amino acid substitutions also encompassnon-naturally-occurring amino acid residues (as defined herein) that aretypically incorporated by chemical peptide synthesis rather than bysynthesis in biological systems. These include peptidomimetics, andother reversed or inverted forms of amino acid moieties.

Naturally-occurring residues can be divided into classes based on commonside chain properties:

(1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr;

(3) acidic: Asp, Glu;

(4) basic: Asn, Gln, His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro; and

(6) aromatic: Trp, Tyr, Phe.

Additional groups of amino acids can also be formulated using theprinciples described in, e.g., Creighton (1984) PROTEINS: STRUCTURE ANDMOLECULAR PROPERTIES (2d Ed. 1993), W. H. Freeman and Company. In someinstances it can be useful to further characterize substitutions basedon two or more of such features (e.g., substitution with a “small polar”residue, such as a Thr residue, can represent a highly conservativesubstitution in an appropriate context).

Conservative substitutions can involve the exchange of a member of oneof these classes for another member of the same class. Non-conservativesubstitutions can involve the exchange of a member of one of theseclasses for a member from another class.

Synthetic, rare, or modified amino acid residues having known similarphysiochemical properties to those of an above-described grouping can beused as a “conservative” substitute for a particular amino acid residuein a sequence. For example, a D-Arg residue may serve as a substitutefor a typical L-Arg residue. It also can be the case that a particularsubstitution can be described in terms of two or more of the abovedescribed classes (e.g., a substitution with a small and hydrophobicresidue means substituting one amino acid with a residue(s) that isfound in both of the above-described classes or other synthetic, rare,or modified residues that are known in the art to have similarphysiochemical properties to such residues meeting both definitions).

Nucleic acid sequences encoding a GDF15 mutant polypeptide providedherein, including those degenerate to SEQ ID NOs: 3, 7, 11 and 15, andthose encoding polypeptide variants of SEQ ID NOs:4, 8 and 12, formother aspects of the instant disclosure.

III.B. Vectors Useful for Expressing GDF15 Polypeptides and ConstructsComprising GDF15, Including Mutant Forms Thereof

In order to express the nucleic acid sequences encoding a polypeptidecomprising a GDF15 region, the appropriate coding sequences, e.g., SEQID NOs:3, 7 and 11, can be cloned into a suitable vector and afterintroduction in a suitable host, the sequence can be expressed toproduce the encoded polypeptide according to standard cloning andexpression techniques, which are known in the art (e.g., as described inSambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: ALaboratory Manual 2nd, ed., Cold Spring Harbor Laboratory, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989). The inventionalso relates to such vectors comprising a nucleic acid sequenceaccording to the invention.

A “vector” refers to a delivery vehicle that (a) promotes the expressionof a polypeptide-encoding nucleic acid sequence; (b) promotes theproduction of the polypeptide therefrom; (c) promotes thetransfection/transformation of target cells therewith; (d) promotes thereplication of the nucleic acid sequence; (e) promotes stability of thenucleic acid; (f) promotes detection of the nucleic acid and/ortransformed/transfected cells; and/or (g) otherwise imparts advantageousbiological and/or physiochemical function to the polypeptide-encodingnucleic acid. A vector can be any suitable vector, includingchromosomal, non-chromosomal, and synthetic nucleic acid vectors (anucleic acid sequence comprising a suitable set of expression controlelements). Examples of such vectors include derivatives of SV40,bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectorsderived from combinations of plasmids and phage DNA, and viral nucleicacid (RNA or DNA) vectors.

A recombinant expression vector can be designed for expression of apolypeptide comprising a GDF15 region in prokaryotic (e.g., E. coli) oreukaryotic cells (e.g., insect cells, using baculovirus expressionvectors, yeast cells, or mammalian cells). Representative host cellsinclude those hosts typically used for cloning and expression, includingEscherichia coli strains TOP10F′, TOP10, DH10B, DH5a, HB101, W3110,BL21(DE3) and BL21 (DE3)pLysS, BLUESCRIPT (Stratagene), mammalian celllines CHO, CHO-K1, HEK293, 293-EBNA pIN vectors (Van Heeke & Schuster,J. Biol. Chem. 264: 5503-5509 (1989); pET vectors (Novagen, MadisonWis.). Alternatively, the recombinant expression vector can betranscribed and translated in vitro, for example using T7 promoterregulatory sequences and T7 polymerase and an in vitro translationsystem. Preferably, the vector contains a promoter upstream of thecloning site containing the nucleic acid sequence encoding thepolypeptide. Examples of promoters, which can be switched on and off,include the lac promoter, the T7 promoter, the trc promoter, the tacpromoter and the trp promoter.

Thus, provided herein are vectors comprising a nucleic acid sequenceencoding a polypeptide comprising a GDF15 region that facilitate theexpression of the polypeptide or construct of interest. In variousembodiments, the vectors comprise an operably linked nucleotide sequencewhich regulates the expression of a polypeptide comprising a GDF15region. A vector can comprise or be associated with any suitablepromoter, enhancer, and other expression-facilitating elements. Examplesof such elements include strong expression promoters (e.g., a human CMVIE promoter/enhancer, an RSV promoter, SV40 promoter, SL3-3 promoter,MMTV promoter, or HIV LTR promoter, EF1alpha promoter, CAG promoter),effective poly (A) termination sequences, an origin of replication forplasmid product in E. coli, an antibiotic resistance gene as aselectable marker, and/or a convenient cloning site (e.g., apolylinker). Vectors also can comprise an inducible promoter as opposedto a constitutive promoter such as CMV IE. In one aspect, a nucleic acidcomprising a sequence encoding a polypeptide comprising a GDF15 regionwhich is operatively linked to a tissue specific promoter which promotesexpression of the sequence in a metabolically-relevant tissue, such asliver or pancreatic tissue is provided.

III.C. Host Cells

In another aspect of the instant disclosure, host cells comprising thenucleic acids and vectors disclosed herein are provided. In variousembodiments, the vector or nucleic acid is integrated into the host cellgenome, which in other embodiments the vector or nucleic acid isextra-chromosomal.

Recombinant cells, such as yeast, bacterial (e.g., E. coli), andmammalian cells (e.g., immortalized mammalian cells) comprising such anucleic acid, vector, or combinations of either or both thereof areprovided. In various embodiments, cells comprising a non-integratednucleic acid, such as a plasmid, cosmid, phagemid, or linear expressionelement, which comprises a sequence coding for expression of apolypeptide comprising a GDF15 region.

A vector comprising a nucleic acid sequence encoding a polypeptidecomprising a GDF15 region can be introduced into a host cell bytransformation or by transfection. Methods of transforming a cell withan expression vector are well known.

A nucleic acid encoding a polypeptide comprising a GDF15 region can bepositioned in and/or delivered to a host cell or host animal via a viralvector. Any suitable viral vector can be used in this capacity. A viralvector can comprise any number of viral polynucleotides, alone or incombination with one or more viral proteins, which facilitate delivery,replication, and/or expression of the nucleic acid of the invention in adesired host cell. The viral vector can be a polynucleotide comprisingall or part of a viral genome, a viral protein/nucleic acid conjugate, avirus-like particle (VLP), or an intact virus particle comprising viralnucleic acids and a nucleic acid encoding a polypeptide comprising aGDF15 region. A viral particle viral vector can comprise a wild-typeviral particle or a modified viral particle. The viral vector can be avector which requires the presence of another vector or wild-type virusfor replication and/or expression (e.g., a viral vector can be ahelper-dependent virus), such as an adenoviral vector amplicon.Typically, such viral vectors consist of a wild-type viral particle, ora viral particle modified in its protein and/or nucleic acid content toincrease transgene capacity or aid in transfection and/or expression ofthe nucleic acid (examples of such vectors include the herpes virus/AAVamplicons). Typically, a viral vector is similar to and/or derived froma virus that normally infects humans. Suitable viral vector particles inthis respect, include, for example, adenoviral vector particles(including any virus of or derived from a virus of the adenoviridae),adeno-associated viral vector particles (AAV vector particles) or otherparvoviruses and parvoviral vector particles, papillomaviral vectorparticles, flaviviral vectors, alphaviral vectors, herpes viral vectors,pox virus vectors, retroviral vectors, including lentiviral vectors.

III.D. Isolation of a GDF15 Polypeptide, Construct Comprising a GDF15Polypeptide or a Mutant Form Thereof

A polypeptide comprising a GDF15 region can be isolated using standardprotein purification methods. A polypeptide comprising a GDF15 regioncan be isolated from a cell that has been engineered to express apolypeptide comprising a GDF15 region, for example a cell that does notnaturally express native GDF15.

Protein purification methods that can be employed to isolate polypeptidecomprising a GDF15 region, as well as associated materials and reagents,are known in the art. Exemplary methods of purifying polypeptidecomprising a GDF15 region are provided in the Examples herein below.Additional purification methods that may be useful for isolatingpolypeptide comprising a GDF15 region can be found in references such asBootcov M R, 1997, Proc. Natl. Acad. Sci. USA 94:11514-9, Fairlie W D,2000, Gene 254: 67-76.

IV. Pharmaceutical Compositions Comprising a GDF15 Polypeptide,Construct Comprising a GDF15 Polypeptide or a Mutant Form Thereof

Pharmaceutical compositions comprising a monomer or multimer comprisinga polypeptide comprising a GDF15 region are provided. Such polypeptidepharmaceutical compositions can comprise a therapeutically effectiveamount of a polypeptide comprising a monomer or multimer comprising apolypeptide comprising a GDF15 region in admixture with apharmaceutically or physiologically acceptable formulation agentselected for suitability with the mode of administration. The term“pharmaceutically acceptable carrier” or “physiologically acceptablecarrier” as used herein refers to one or more formulation agentssuitable for accomplishing or enhancing the delivery of a monomer ormultimer comprising a polypeptide comprising a GDF15 region into thebody of a human or non-human subject. The term includes any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Examples of pharmaceutically acceptablecarriers include one or more of water, saline, phosphate bufferedsaline, dextrose, glycerol, ethanol and the like, as well ascombinations thereof. In some cases it will be preferable to includeisotonic agents, for example, sugars, polyalcohols such as mannitol,sorbitol, or sodium chloride in a pharmaceutical composition.Pharmaceutically acceptable substances such as wetting or minor amountsof auxiliary substances such as wetting or emulsifying agents,preservatives or buffers, which enhance the shelf life or effectivenessof the monomer or multimer comprising a polypeptide comprising a GDF15region can also act as, or form a component of, a carrier. Acceptablepharmaceutically acceptable carriers are preferably nontoxic torecipients at the dosages and concentrations employed.

A pharmaceutical composition can contain formulation agent(s) formodifying, maintaining, or preserving, for example, the pH, osmolarity,viscosity, clarity, color, isotonicity, odor, sterility, stability, rateof dissolution or release, adsorption, or penetration of thecomposition. Suitable formulation agents include, but are not limitedto, amino acids (such as glycine, glutamine, asparagine, arginine, orlysine), antimicrobials, antioxidants (such as ascorbic acid, sodiumsulfite, or sodium hydrogen-sulfite), buffers (such as borate,bicarbonate, Tris-HCl, citrates, phosphates, or other organic acids),bulking agents (such as mannitol or glycine), chelating agents (such asethylenediamine tetraacetic acid (EDTA)), complexing agents (such ascaffeine, polyvinylpyrrolidone, beta-cyclodextrin, orhydroxypropyl-beta-cyclodextrin), fillers, monosaccharides,disaccharides, and other carbohydrates (such as glucose, mannose, ordextrins), proteins (such as free serum albumin, gelatin, orimmunoglobulins), coloring, flavoring and diluting agents, emulsifyingagents, hydrophilic polymers (such as polyvinylpyrrolidone), lowmolecular weight polypeptides, salt-forming counterions (such assodium), preservatives (such as benzalkonium chloride, benzoic acid,salicylic acid, thimerosal, phenethyl alcohol, methylparaben,propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide),solvents (such as glycerin, propylene glycol, or polyethylene glycol),sugar alcohols (such as mannitol or sorbitol), suspending agents,surfactants or wetting agents (such as pluronics; PEG; sorbitan esters;polysorbates such as Polysorbate 20 or Polysorbate 80; Triton;tromethamine; lecithin; cholesterol or tyloxapal), stability enhancingagents (such as sucrose or sorbitol), tonicity enhancing agents (such asalkali metal halides—preferably sodium or potassium chloride—or mannitolsorbitol), delivery vehicles, diluents, excipients and/or pharmaceuticaladjuvants (see, e.g., REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY,19th edition, (1995); Berge et al., J. Pharm. Sci., 6661), 1-19 (1977).Additional relevant principles, methods, and agents are described in,e.g., Lieberman et al., PHARMACEUTICAL DOSAGE FORMS-DISPERSE SYSTEMS(2nd ed., vol. 3, 1998); Ansel et al., PHARMACEUTICAL DOSAGE FORMS &DRUG DELIVERY SYSTEMS (7th ed. 2000); Martindale, THE EXTRA PHARMACOPEIA(31st edition), Remington's PHARMACEUTICAL SCIENCES (16th-20^(th) andsubsequent editions); The Pharmacological Basis Of Therapeutics, Goodmanand Gilman, Eds. (9th ed.—1996); Wilson and Gisvolds' TEXTBOOK OFORGANIC MEDICINAL AND PHARMACEUTICAL CHEMISTRY, Delgado and Remers, Eds.(10th ed., 1998). Principles of formulating pharmaceutically acceptablecompositions also are described in, e.g., Aulton, PHARMACEUTICS: THESCIENCE OF DOSAGE FORM DESIGN, Churchill Livingstone (New York) (1988),EXTEMPORANEOUS ORAL LIQUID DOSAGE PREPARATIONS, CSHP (1998)).

The optimal pharmaceutical composition will be determined by a skilledartisan depending upon, for example, the intended route ofadministration, delivery format, and desired dosage (see, e.g.,Remington's PHARMACEUTICAL SCIENCES, supra). Such compositions caninfluence the physical state, stability, rate of in vivo release, andrate of in vivo clearance of a GDF15 polypeptide, construct comprising aGDF15 polypeptide or a mutant form thereof.

The primary vehicle or carrier in a pharmaceutical composition can beeither aqueous or non-aqueous in nature. For example, a suitable vehicleor carrier for injection can be water, physiological saline solution, orartificial cerebrospinal fluid, possibly supplemented with othermaterials common in compositions for parenteral administration. Neutralbuffered saline or saline mixed with free serum albumin are furtherexemplary vehicles. Other exemplary pharmaceutical compositions compriseTris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5,which can further include sorbitol or a suitable substitute. In oneembodiment of the present invention, compositions comprising a GDF15polypeptide, construct comprising a GDF15 polypeptide or a mutant formthereof can be prepared for storage by mixing the selected compositionhaving the desired degree of purity with optional formulation agents(Remington's PHARMACEUTICAL SCIENCES, supra) in the form of alyophilized cake or an aqueous solution. Furthermore, a productcomprising a monomer or multimer comprising a polypeptide comprising aGDF15 region can be formulated as a lyophilizate using appropriateexcipients such as sucrose.

The polypeptide pharmaceutical compositions can be selected forparenteral delivery. Alternatively, the compositions can be selected forinhalation or for delivery through the digestive tract, such as orally.The preparation of such pharmaceutically acceptable compositions iswithin the skill of the art.

The formulation components are present in concentrations that areacceptable to the site of administration. For example, buffers are usedto maintain the composition at physiological pH or at a slightly lowerpH, typically within a pH range of from about 5 to about 8.

When parenteral administration is contemplated, the therapeuticcompositions for use in this invention can be in the form of apyrogen-free, parenterally acceptable, aqueous solution comprising adesired GDF15 polypeptide, construct comprising a GDF15 polypeptide or amutant form thereof, in a pharmaceutically acceptable vehicle. Aparticularly suitable vehicle for parenteral injection is steriledistilled water in which a GDF15 polypeptide, construct comprising amonomer or multimer comprising a polypeptide comprising a GDF15 regionis formulated as a sterile, isotonic solution, properly preserved. Yetanother preparation can involve the formulation of the desired moleculewith an agent, such as injectable microspheres, bio-erodible particles,polymeric compounds (such as polylactic acid or polyglycolic acid),beads, or liposomes, that provides for the controlled or sustainedrelease of the product which can then be delivered via a depotinjection. Hyaluronic acid can also be used, and this can have theeffect of promoting sustained duration in the circulation. Othersuitable means for the introduction of the desired molecule includeimplantable drug delivery devices.

In one embodiment, a pharmaceutical composition can be formulated forinhalation. For example, a monomer or multimer comprising a polypeptidecomprising a GDF15 region can be formulated as a dry powder forinhalation. Inhalation solutions comprising a monomer or multimercomprising a polypeptide comprising a GDF15 region can also beformulated with a propellant for aerosol delivery. In yet anotherembodiment, solutions can be nebulized. Pulmonary administration isfurther described in International Publication No. WO 94/20069, whichdescribes the pulmonary delivery of chemically modified proteins.

It is also contemplated that certain formulations can be administeredorally. In one embodiment of the present invention, a monomer ormultimer comprising a polypeptide comprising a GDF15 region that isadministered in this fashion can be formulated with or without thosecarriers customarily used in the compounding of solid dosage forms suchas tablets and capsules. For example, a capsule can be designed torelease the active portion of the formulation at the point in thegastrointestinal tract when bioavailability is maximized andpre-systemic degradation is minimized Additional agents can be includedto facilitate absorption of a monomer or multimer comprising apolypeptide comprising a GDF15 region. Diluents, flavorings, low meltingpoint waxes, vegetable oils, lubricants, suspending agents, tabletdisintegrating agents, and binders can also be employed.

Another pharmaceutical composition can involve an effective quantity ofa monomer or multimer comprising a polypeptide comprising a GDF15 regionin a mixture with nontoxic excipients that are suitable for themanufacture of tablets. By dissolving the tablets in sterile water, oranother appropriate vehicle, solutions can be prepared in unit-doseform. Suitable excipients include, but are not limited to, inertdiluents, such as calcium carbonate, sodium carbonate or bicarbonate,lactose, or calcium phosphate; or binding agents, such as starch,gelatin, or acacia; or lubricating agents such as magnesium stearate,stearic acid, or talc.

Additional pharmaceutical compositions comprising a monomer or multimercomprising a polypeptide comprising a GDF15 region will be evident tothose skilled in the art, including formulations comprising a monomer ormultimer comprising a polypeptide comprising a GDF15 region, insustained- or controlled-delivery formulations. Techniques forformulating a variety of other sustained- or controlled-delivery means,such as liposome carriers, bio-erodible microparticles or porous beadsand depot injections, are also known to those skilled in the art (see,e.g., International Publication No. WO 93/15722, which describes thecontrolled release of porous polymeric microparticles for the deliveryof pharmaceutical compositions, and Wischke & Schwendeman, 2008, Int. J.Pharm. 364: 298-327, and Freiberg & Zhu, 2004, Int. J. Pharm. 282: 1-18,which discuss microsphere/microparticle preparation and use). Asdescribed herein, a hydrogel is an example of a sustained- orcontrolled-delivery formulation.

Additional examples of sustained-release preparations includesemipermeable polymer matrices in the form of shaped articles, e.g.films, or microcapsules. Sustained release matrices can includepolyesters, hydrogels, polylactides (U.S. Pat. No. 3,773,919 andEuropean Patent No. 0 058 481), copolymers of L-glutamic acid and gammaethyl-L-glutamate (Sidman et al., 1983, Biopolymers 22: 547-56),poly(2-hydroxyethyl-methacrylate) (Langer et al., 1981, J. Biomed.Mater. Res. 15: 167-277 and Langer, 1982, Chem. Tech. 12: 98-105),ethylene vinyl acetate (Langer et al., supra) orpoly-D(−)-3-hydroxybutyric acid (European Patent No. 0 133 988).Sustained-release compositions can also include liposomes, which can beprepared by any of several methods known in the art. See, e.g., Epsteinet al., 1985, Proc. Natl. Acad. Sci. U.S.A. 82: 3688-92; and EuropeanPatent Nos. 0 036 676, 0 088 046, and 0 143 949.

A pharmaceutical composition comprising a monomer or multimer comprisinga polypeptide comprising a GDF15 region which is to be used for in vivoadministration typically should be sterile. This can be accomplished byfiltration through sterile filtration membranes. Where the compositionis lyophilized, sterilization using this method can be conducted eitherprior to, or following, lyophilization and reconstitution. Thecomposition for parenteral administration can be stored in lyophilizedform or in a solution. In addition, parenteral compositions generallyare placed into a container having a sterile access port, for example,an intravenous solution bag or vial having a stopper pierceable by ahypodermic injection needle.

Once the pharmaceutical composition has been formulated, it can bestored in sterile vials as a solution, suspension, gel, emulsion, solid,or as a dehydrated or lyophilized powder. Such formulations can bestored either in a ready-to-use form or in a form (e.g., lyophilized)requiring reconstitution prior to administration.

In a specific embodiment, the present invention is directed to kits forproducing a single-dose administration unit. The kits can each containboth a first container having a dried protein and a second containerhaving an aqueous formulation. Also included within the scope of thisinvention are kits containing single and multi-chambered pre-filledsyringes (e.g., liquid syringes and lyosyringes).

The effective amount of pharmaceutical composition comprising a monomeror multimer comprising a polypeptide comprising a GDF15 region which isto be employed therapeutically will depend, for example, upon thetherapeutic context and objectives. One skilled in the art willappreciate that the appropriate dosage levels for treatment will thusvary depending, in part, upon the molecule delivered, the indication forwhich a monomer or multimer comprising a polypeptide comprising a GDF15region is being used, the route of administration, and the size (bodyweight, body surface, or organ size) and condition (the age and generalhealth) of the patient. Accordingly, the clinician can titer the dosageand modify the route of administration to obtain the optimal therapeuticeffect. A typical dosage can range from about 0.1 μg/kg to up to about100 mg/kg or more, depending on the factors mentioned above. In otherembodiments, the dosage can range from 0.1 μg/kg up to about 100 mg/kg;or 1 μg/kg up to about 100 mg/kg; or 5 μg/kg, 10 μg/kg, 15 μg/kg, 20μg/kg, 25 μg/kg, 30 μg/kg, 35 μg/kg, 40 μg/kg, 45 μg/kg, 50 μg/kg, 55μg/kg, 60 μg/kg, 65 μg/kg, 70 μg/kg, 75 μg/kg, 100 μg/kg, 200 μg/kg orup to about 10 mg/kg.

The frequency of dosing will depend upon the pharmacokinetic parametersof the a monomer or multimer comprising a polypeptide comprising a GDF15region in the formulation being used. Typically, a clinician willadminister the composition until a dosage is reached that achieves thedesired effect. The composition can therefore be administered as asingle dose, as two or more doses (which may or may not contain the sameamount of the desired molecule) over time, or as a continuous infusionvia an implantation device or catheter. Further refinement of theappropriate dosage is routinely made by those of ordinary skill in theart and is within the ambit of tasks routinely performed by them.Appropriate dosages can be ascertained through use of appropriatedose-response data.

The route of administration of the pharmaceutical composition is inaccord with known methods, e.g., orally; through injection byintravenous, intraperitoneal, intracerebral (intraparenchymal),intracerebroventricular, intramuscular, intraocular, intraarterial,intraportal, or intralesional routes; by sustained release systems(which may also be injected); or by implantation devices. Where desired,the compositions can be administered by bolus injection or continuouslyby infusion, or by implantation device.

Alternatively or additionally, the composition can be administeredlocally via implantation of a membrane, sponge, or other appropriatematerial onto which the desired molecule has been absorbed orencapsulated. Where an implantation device is used, the device can beimplanted into any suitable tissue or organ, and delivery of the desiredmolecule can be via diffusion, timed-release bolus, or continuousadministration.

In order to deliver drug, e.g., a monomer or multimer comprising apolypeptide comprising a GDF15 region, at a predetermined rate such thatthe drug concentration can be maintained at a desired therapeuticallyeffective level over an extended period, a variety of differentapproaches can be employed. In one example, a hydrogel comprising apolymer such as a gelatin (e.g., bovine gelatin, human gelatin, orgelatin from another source) or a naturally-occurring or a syntheticallygenerated polymer can be employed. Any percentage of polymer (e.g.,gelatin) can be employed in a hydrogel, such as 5, 10, 15 or 20%. Theselection of an appropriate concentration can depend on a variety offactors, such as the therapeutic profile desired and the pharmacokineticprofile of the therapeutic molecule.

Examples of polymers that can be incorporated into a hydrogel includepolyethylene glycol (“PEG”), polyethylene oxide, polyethyleneoxide-co-polypropylene oxide, co-polyethylene oxide block or randomcopolymers, polyvinyl alcohol, poly(vinyl pyrrolidinone), poly(aminoacids), dextran, heparin, polysaccharides, polyethers and the like.

Another factor that can be considered when generating a hydrogelformulation is the degree of crosslinking in the hydrogel and thecrosslinking agent. In one embodiment, cross-linking can be achieved viaa methacrylation reaction involving methacrylic anhydride. In somesituations, a high degree of cross-linking may be desirable while inother situations a lower degree of crosslinking is preferred. In somecases a higher degree of crosslinking provides a longer sustainedrelease. A higher degree of crosslinking may provide a firmer hydrogeland a longer period over which drug is delivered.

Any ratio of polymer to crosslinking agent (e.g., methacrylic anhydride)can be employed to generate a hydrogel with desired properties. Forexample, the ratio of polymer to crosslinker can be, e.g., 8:1, 16:1,24:1, or 32:1. For example, when the hydrogel polymer is gelatin and thecrosslinker is methacrylate, ratios of 8:1, 16:1, 24:1, or 32:1methacrylic anhydride:gelatin can be employed.

V. Therapeutic Uses of a GDF15 Polypeptide, Construct Comprising a GDF15Polypeptide or a Mutant Form Thereof

A monomer or multimer comprising a polypeptide comprising a GDF15 regioncan be used to treat, diagnose or ameliorate, a metabolic condition ordisorder. In one embodiment, the metabolic disorder to be treated isdiabetes, e.g., type 2 diabetes. In another embodiment, the metaboliccondition or disorder is obesity. In other embodiments the metaboliccondition or disorder is dyslipidemia, elevated glucose levels, elevatedinsulin levels or diabetic nephropathy. For example, a metaboliccondition or disorder that can be treated or ameliorated using a monomeror multimer comprising a polypeptide comprising a GDF15 region includesa state in which a human subject has a fasting blood glucose level of125 mg/dL or greater, for example 130, 135, 140, 145, 150, 155, 160,165, 170, 175, 180, 185, 190, 195, 200 or greater than 200 mg/dL. Bloodglucose levels can be determined in the fed or fasted state, or atrandom. The metabolic condition or disorder can also comprise acondition in which a subject is at increased risk of developing ametabolic condition. For a human subject, such conditions include afasting blood glucose level of 100 mg/dL. Conditions that can be treatedusing a pharmaceutical composition comprising a GDF15 mutant polypeptidecan also be found in the American Diabetes Association Standards ofMedical Care in Diabetes Care-2011, American Diabetes Association,Diabetes Care Vol. 34, No. Supplement 1, S11-S61, 2010.

In application, a metabolic disorder or condition, such as Type 2diabetes, elevated glucose levels, elevated insulin levels,dyslipidemia, obesity or diabetic nephropathy, can be treated byadministering a therapeutically effective dose of a GDF15 polypeptide,construct comprising a monomer or multimer comprising a polypeptidecomprising a GDF15 region to a patient in need thereof. Theadministration can be performed as described herein, such as by IVinjection, intraperitoneal (IP) injection, subcutaneous injection,intramuscular injection, or orally in the form of a tablet or liquidformation. In some situations, a therapeutically effective or preferreddose of a monomer or multimer comprising a polypeptide comprising aGDF15 region can be determined by a clinician. A therapeuticallyeffective dose of a monomer or multimer comprising a polypeptidecomprising a GDF15 region will depend, inter alia, upon theadministration schedule, the unit dose of agent administered, whetherthe a monomer or multimer comprising a polypeptide comprising a GDF15region is administered in combination with other therapeutic agents, theimmune status and the health of the recipient. The term “therapeuticallyeffective dose,” as used herein, means an amount of a monomer ormultimer comprising a polypeptide comprising a GDF15 region that elicitsa biological or medicinal response in a tissue system, animal, or humanbeing sought by a researcher, medical doctor, or other clinician, whichincludes alleviation or amelioration of the symptoms of the disease ordisorder being treated, i.e., an amount of a monomer or multimercomprising a polypeptide comprising a GDF15 region that supports anobservable level of one or more desired biological or medicinalresponse, for example, lowering blood glucose, insulin, triglyceride, orcholesterol levels; reducing body weight; or improving glucosetolerance, energy expenditure, or insulin sensitivity.

It is noted that a therapeutically effective dose of a monomer ormultimer comprising a polypeptide comprising a GDF15 region can alsovary with the desired result. Thus, for example, in situations in whicha lower level of blood glucose is indicated a dose of a monomer ormultimer comprising a polypeptide comprising a GDF15 region will becorrespondingly higher than a dose in which a comparatively lower levelof blood glucose is desired. Conversely, in situations in which a higherlevel of blood glucose is indicated at a dose of a monomer or multimercomprising a polypeptide comprising a GDF15 region will becorrespondingly lower than a dose in which a comparatively higher levelof blood glucose is desired.

In various embodiments, a subject is a human having a blood glucoselevel of 100 mg/dL or greater can be treated with a monomer or multimercomprising a polypeptide comprising a GDF15 region.

In one embodiment, a method of the instant disclosure comprises firstmeasuring a baseline level of one or more metabolically-relevantcompounds such as glucose, insulin, cholesterol, lipid in a subject. Apharmaceutical composition comprising a monomer or multimer comprising apolypeptide comprising a GDF15 region is then administered to thesubject. After a desired period of time, the level of the one or moremetabolically-relevant compounds (e.g., blood glucose, insulin,cholesterol, lipid) in the subject is again measured. The two levels canthen be compared in order to determine the relative change in themetabolically-relevant compound in the subject. Depending on the outcomeof that comparison another dose of the pharmaceutical compositioncomprising a monomer or multimer comprising a polypeptide comprising aGDF15 region can be administered to achieve a desired level of one ormore metabolically-relevant compound.

It is noted that a pharmaceutical composition comprising a monomer ormultimer comprising a polypeptide comprising a GDF15 region can beco-administered with another compound. The identity and properties ofcompound co-administered with the a monomer or multimer comprising apolypeptide comprising a GDF15 region will depend on the nature of thecondition to be treated or ameliorated. A non-limiting list of examplesof compounds that can be administered in combination with apharmaceutical composition comprising a monomer or multimer comprising apolypeptide comprising a GDF15 region include rosiglitizone,pioglitizone, repaglinide, nateglitinide, metformin, exenatide,stiagliptin, pramlintide, glipizide, glimeprirideacarbose, and miglitol.

VI. Kits

Also provided are kits for practicing the disclosed methods. Such kitscan comprise a pharmaceutical composition such as those describedherein, including nucleic acids encoding the peptides or proteinsprovided herein, vectors and cells comprising such nucleic acids, andpharmaceutical compositions comprising such nucleic acid-containingcompounds, which can be provided in a sterile container. Optionally,instructions on how to employ the provided pharmaceutical composition inthe treatment of a metabolic disorder can also be included or be madeavailable to a patient or a medical service provider.

In one aspect, a kit comprises (a) a pharmaceutical compositioncomprising a therapeutically effective amount of a monomer or multimercomprising a polypeptide comprising a GDF15 region; and (b) one or morecontainers for the pharmaceutical composition. Such a kit can alsocomprise instructions for the use thereof; the instructions can betailored to the precise metabolic disorder being treated. Theinstructions can describe the use and nature of the materials providedin the kit. In certain embodiments, kits include instructions for apatient to carry out administration to treat a metabolic disorder, suchas elevated glucose levels, elevated insulin levels, obesity, type 2diabetes, dyslipidemia or diabetic nephropathy.

Instructions can be printed on a substrate, such as paper or plastic,etc, and can be present in the kits as a package insert, in the labelingof the container of the kit or components thereof (e.g., associated withthe packaging), etc. In other embodiments, the instructions are presentas an electronic storage data file present on a suitable computerreadable storage medium, e.g. CD-ROM, diskette, etc. In yet otherembodiments, the actual instructions are not present in the kit, butmeans for obtaining the instructions from a remote source, such as overthe internet, are provided. An example of this embodiment is a kit thatincludes a web address where the instructions can be viewed and/or fromwhich the instructions can be downloaded.

Often it will be desirable that some or all components of a kit arepackaged in suitable packaging to maintain sterility. The components ofa kit can be packaged in a kit containment element to make a single,easily handled unit, where the kit containment element, e.g., box oranalogous structure, may or may not be an airtight container, e.g., tofurther preserve the sterility of some or all of the components of thekit.

EXAMPLES

The following examples, including the experiments conducted and resultsachieved, are provided for illustrative purposes only and are not to beconstrued as limiting the present invention.

Example 1 Preparation of Fc-GDF15 Molecules

GDF15 fusions with knob/holeFc, HemiFc, charged pair (delHinge) Fc andcharged pair (delHinge) cysteine clamp Fc sequences were stablyexpressed in serum free, suspension adapted CHO-K1 cell line. GDF15-Fcmolecules were cloned into a stable expression vector containingpuromycin resistance while the Fc chains were cloned into a hygromycincontaining expression vector (Selexis, Inc.). The plasmids weretransfected at a 1:1 ratio using lipofectamine LTX and cells wereselected 2 days post transfection in a proprietary growth mediacontaining 10 ug/mL puromycin and 600 ug/mL hygromycin. Media wasexchanged 2 times per week during selection. When cells reached about90% viability, they were scaled up for a fedbatch production run. Cellswere seeded at 1e6/mL in a proprietary production media and fed on days3, 6, and 8. The conditioned medium (CM) produced by the cells washarvested on day 10 and clarified. Endpoint viabilities typically wereabove 90%.

The Fc-GDF15 clarified, conditioned media was purified using a two-stepchromatography procedure. Approximately 5 L of the CM was applieddirectly to a GE MabSelect SuRe column that had previously beenequilibrated with Dulbecco's Phosphate Buffered Saline (PBS). The boundprotein underwent three wash steps: first, 3 column volumes (CV) of PBS;next, 1 CV of 20 mM Tris, 100 mM sodium chloride, pH 7.4; and finally, 3CV of 500 mM L-arginine, pH 7.5. These wash steps remove unbound orlightly bound media components and host cell impurities. The column wasthen re-equilibrated with 5 CV of 20 mM Tris, 100 mM sodium chloride atpH 7.4 which brings the UV absorbance back to baseline. The desiredprotein was eluted with 100 mM acetic acid at pH 3.6 and collected inbulk. The protein pool was quickly titrated to within a pH range of 5.0to 5.5 with 1 M Tris-HCl, pH 9.2.

The pH adjusted protein pool was next loaded onto a GE SP Sepharose HPcolumn that had been previously equilibrated with 20 mM MES at pH 6.0.The bound protein was then washed with 5 CV of equilibration buffer, andfinally eluted over a 20 CV, 0 to 50% linear gradient from 0 to 400 mMsodium chloride in 20 mM MES at pH 6.0. Fractions were collected duringthe elution and analyzed by analytical size-exclusion chromatography(Superdex 200) to determine the appropriate fractions to pool for ahomogeneous product. The SP HP chromatography removes product-relatedimpurities such as free Fc, clipped species, and Fc-GDF15 multimers.

The SP HP pool was then buffer exchanged into 10 mM sodium acetate, 5%proline, pH 5.2 by dialysis. It was concentrated to approximately 15mg/ml using the Sartorius Vivaspin 20 Ten kilo-Dalton molecular weightcut-off centrifugal device. Finally, it was sterile filtered and theresulting solution containing the purified Fc-GDF15 molecules is storedat 5° C. Final products were assessed for identity and purity using massspectral analysis, sodium dodecyl sulfate polyacrylamide electrophoresisand size exclusion high performance liquid chromatography.

The purification method described above was employed to purifyDhMonoFc-GDF15 fusion proteins. However, it was found that the additionof the H6D mutation to the DhMonoFc-GDF15 caused soluble aggregates toform in the SP elution. Therefore, the purification of theDhMonoFc-GFF15(H6D) included an additional SEC step (Superdex 200 with20 mM phosphate, 250 mM NaCl, pH 6.8), followed by loading onQ-sepharose HP and eluting with a gradient from 0 to 0.6M NaCl in 20 mMtris, pH 8.5.

Example 2 Preparation of GDF15-HSA and DhMonoFc Molecules

GDF15 fusions with HSA and DhMonoFc sequences were stably expressed inCHO-S cells (Invitrogen). For each of the constructs producinghomodimers, the coding sequence was cloned into stable expression vectorcontaining puromycin resistance (Selexis, Inc.). In the case of theHSA-(G₄S)₄-GDF15:GDF15 heterodimer, the HSA-(G₄S)₄-GDF15 fusion sequencewas cloned into an expression vector containing puromycin resistance andthe GDF15 sequence was cloned into an expression vector containinghygromycin resistance. CHO-S parental cells were maintained in CD-CHOmedium (Invitrogen) supplemented with 8 mM L-glutamine and weretransfected with 4 μg of plasmid DNA using a Lipofectamine LTXtransfection kit (Invitrogen) according to the manufacturer'sinstructions. In the case of the HSA-(G₄S)₄-GDF15:GDF15 heterodimer, thetwo plasmids were mixed at a 1:1 ratio prior to transfection. Stablecell lines were selected using 10 μg/mL of puromycin (homodimers) or 10μg/mL of puromycin plus 400 μg/mL hygromycin (heterodimer). Uponrecovery, which was defined as >90% viability using a Vi-Cell counter(Beckman Coulter), the stable CHO-S cell lines were expanded and used toseed either batch productions in shake flasks or fedbatch productions inWAVE bioreactors (GE Healthcare). Both processes were seeded at 1e6viable cells/mL in production medium. Batch productions were harvestedby centrifugation on day 6, while fedbatch productions were fed on days3, 6, and 8. The CM produced by the cells was harvested bycentrifugation on day 10 and clarified.

The HSA-GDF15 fusion proteins were purified from clarified conditionedmedia using two chromatographic steps. The clarified conditioned mediacontaining the HSA-GDF15 fusion protein was applied to a Cibracon BlueSepharose HP column that is equilibrated with 20 mM Phosphate, 150 mMNaCl pH 7.4. The column was next washed with equilibration buffer untila baseline ultraviolet (UV) level is obtained. Product and contaminantsare eluted by a 20 mM Phosphate, 2M NaCl buffer and the elutions werecollected and subsequently assayed by Coomasie-stained SDS-PAGE (sodiumdodecyl sulfate polyacrylamide gel electrophoresis) to identify whicheluate fractions contained a polypeptide that migrates at the predictedmolecular weight of HSA-GDF15 fusion protein. Following the BlueSepharose step, the pooled fractions containing product were dialyzedversus 10 mM tris, pH 8.0. The dialysis step allowed for the binding ofHSA-GDF15 fusion protein when applied to anion exchange chromatographicresin. The final chromatography step was Q-Sepharose HP which applies alinear gradient (0 to 0.6M NaCl in 10 mM tris pH 8.0) to elute the boundfusion protein. The elution from the Q-Sepharose HP was collected asfractions and then assayed by SDS-PAGE and analytical size exclusionchromatography to determine the appropriate fractions to pool. LCMS andSDS-PAGE were run to confirm the identity of each protein. The resultingpool was buffer exchanged by dialysis into 10 mM Sodium Acetate, 9%sucrose pH 4.5, sterile filtered, and finally stored at 5 C or frozen.

DhMonoFc-GDF15 fusion proteins were purified as set forth above inExample 2 for other Fc-GDF15 fusion proteins.

Example 3 Suppression of Food Intake in Hyperphagic Ob/Ob Mice by FcFusion GDF15 Polypeptides and HSA Fusion GDF15 Polypeptides

GDF15 reduces food intake in hyperphagic ob/ob mice, and a food intakeassay was used to evaluate efficacy of different forms of GDF15 analogs.As the half-life of human GDF15 polypeptide in mouse was observed to beapproximately 3 hours, an Fc fusion strategy was used to extend proteinhalf-life. Various multimers comprising a polypeptide comprising a GDF15region were generated and analyzed for in vivo activity, by introducingthe multimer into hyperphagic leptin-deficient ob/ob mice, and measuringthe ability of a particular multimer comprising a polypeptide comprisinga GDF15 region to suppress food intake in these animals. The multimercomprising a polypeptide comprising a GDF15 region to be tested wasinjected subcutaneously into a 7-8 week old ob/ob mouse (JacksonLaboratory) between 4-5 pm on day 0. Animals were transferred afterinjection to cages where food had been premeasured, and food intake wasmeasured between 9-10 AM the next day.

The results of representative experiments are provided in FIGS. 6-53.These experiments demonstrate that the described multimers comprisingGDF15 regions exhibit a decrease in food intake in ob/ob mice, withgreater potency than those of native mature hGDF15 homodimer.

Example 4 Chronic Efficacy of GDF15 Constructs in DIO Mice

Certain multimers comprising GDF15 regions are administered chronicallyand subcutaneously into DIO mice, once per week. The constructsdemonstrate efficacy in improving various metabolic parameters,including body weight, blood glucose levels and glucose tolerance, seruminsulin levels, serum cholesterol levels, serum triglyceride levels andoral lipid tolerance.

Example 5 In Vivo Activity of GDF15 Constructs

Male C57Bl/6 were fed a 60% high fat diet for 15 weeks and divided intodifferent treatment groups for each group to have the same pretreatmentbody weight, glucose, insulin, triglyceride and cholesterol levels.Animals were subcutaneously dosed with proteins or vehicle buffer weeklyfor 5 weeks. Three different dose levels were selected for the proteins:10, 1, 0.1 nmol/kg, which are equivalent to 1.25, 0.125, 0.0125 mg/kg.Studies were carried for 5 weeks, with the last dose on day 28.

Body weight was measured weekly during the 5 weeks of treatment and drugwashout. One oral glucose tolerance tests (OGTT) was performed 2 weeksafter the first protein injection in animals fasted for 4 hours. Anotheroral glucose tolerance tests (OGTT) was performed 5 weeks after thefirst protein injection in animals fasted for 16 hours. In OGTT, animalswere orally administered with 2 g/kg glucose solution, and glucoselevels were measured by AlphaTRAK glucometer (Abbott) at 0, 15, 30, 60,120 min. Area under the curve (AUC) of the glucose levels during theOGTT were calculated to compare glucose tolerance of different treatmentgroups. Serum samples were collected at 3 weeks after first proteininjection and used to measure insulin, triglyceride and cholesterollevels, as well as the levels of test articles. Insulin levels weremeasured using immunoassay kit (Alpco). Triglyceride and cholesterollevels were measured using enzymatic assays (Wako).

The results are shown in FIGS. 54-59 (asterisks indicate statisticalsignificance). These experiments demonstrate that the describedmultimers comprising GDF15 regions reduce AUC of the glucose levelsduring the OGTT (FIGS. 55 and 59), reduce body weight (FIG. 54) reduceinsulin levels (FIG. 56), reduce cholesterol (FIG. 58) and reducetriglycerides (FIG. 57).

Example 6 Thermal Stability of GDF15 Constructs

The thermal stability of the selected GDF15 constructs was assessed bydifferential scanning calorimetry on a MicroCal Capillary VP-DSC systemin which temperature differences between the reference and sample cellare continuously measured, and calibrated to power units. This datachannel is referred to as the DP signal, or the differential powerbetween the reference and sample cell. The unfolding of a proteinmolecule appears as an endothermic transition on the DSC thermogram andcan characterized by the thermal transition midpoints (Tm). The sampleswere heated from 10° C. to 100° C. at a heating rate of 60° C./hour. Thepre-scan time was 15 minutes and the filtering period was 10 seconds.The concentrations used in the DSC experiments were around 1.0 mg/mL.The data analysis for baseline correction and determination of Tm valueswas done using MicroCal Origin 7 software.

In particular, a dimer of DhCpmFc(−)-GDF15(N3D):DhCpmFc(+) was comparedto a dimer of Dh3CpmFc(−)-GDF15(N3D):DhCpmFc(+); a dimer ofDhCpmFc(−)-GDF15(Ndel3):DhCpmFc(+) was compared to a dimer ofDh3CpmFc(−)-GDF15(Ndel3):DhCpmFc(+); a dimer ofDhCpmFc(−)(Y349C)-GDF15(N3D):DhCpmFc(+)(S354C) was compared to a dimerof Dh3CpmFc(−)(Y349C)-GDF15(N3D):DhCpmFc(+)(S354C); and a dimer ofDhCpmFc(−)(Y349C)-GDF15(Ndel3):DhCpmFc(+)(S354C) was compared to a dimerof Dh3CpmFc(−)(Y349C)-GDF15(Ndel3):DhCpmFc(+)(S354C). The results areshown in FIG. 61. These experiments demonstrate that the “Dh3CpmFc”domains confer greater stability than the corresponding “DhCpmFc”domains.

Example 7 Fcγ Receptor Binding Analysis

Selected GDF15 constructs were analyzed for their binding activity toFcγ receptors on BIA3000. Each Fcγ receptor was captured on anti-hisantibody coated CM5 surface (captured RL˜200 RU). The GDF15 constructswere diluted to 250 nM in sample buffer (0.1 mg/ml BSA, 0.005% P20,PBS). Each GDF15 construct was injected over anti-his antibody capturedFcγ receptor surfaces at 50 μL/min for 3 minutes. After a 5-minutedissociation in instrument running buffer (0.005% P20 in PBS), each Fcγreceptor surface was regenerated by an injection of 8 mM Glycine, pH1.5,1M NaCl for 30 seconds, followed by an injection of 10 mM Glycine, pH1.5for 30 seconds. The resulting sensorgrams were analyzed using BIAcoreBIAEvaluation (v. 4.1). The binding response in the unit of RU was readat 10 seconds before end of injection.

In particular, FcγRI, FcγRIIIA and FcγRIIA were determined with respectto a dimer of DhCpmFc(−)-GDF15(Ndel3):DhCpmFc(+), a dimer ofDhCpmFc(−)(Y349C)-GDF15(Ndel3):DhCpmFc(+)(S354C); a dimer ofDh3CpmFc(−)-GDF15(Ndel3); a dimer ofDh3CpmFc(−)(Y349C)-GDF15(Ndel3)-Dh3CpmFc(+)(S354C); a dimer ofDh3CpmFc(−)-GDF15(N3D); and a dimer ofDh3CpmFc(−)(Y349C)-GDF15(N3D):Dh3CpmFc(+)(S354C). The results are shownin FIG. 60. These experiments demonstrate that the “Dh3CpmFc” domainsessentially eliminate FcγRI, FcγRIIIA and FcγRIIA binding.

While the present invention has been described in terms of variousembodiments, it is understood that variations and modifications willoccur to those skilled in the art. Therefore, it is intended that theappended claims cover all such equivalent variations that come withinthe scope of the invention as claimed. In addition, the section headingsused herein are for organizational purposes only and are not to beconstrued as limiting the subject matter described.

All references cited in this application are expressly incorporated byreference herein for any purpose.

What is claimed is:
 1. A fusion protein comprising a GDF15 region and anFc domain, wherein the Fc domain has the amino acid sequence of SEQ IDNO: 220 or SEQ ID NO: 220 with a tyrosine to cysteine mutation atposition
 113. 2. The fusion protein of claim 1, wherein the GDF15 regioncomprises the amino acid sequence of SEQ ID NO: 4, 8, 12, 25, 52 or 55.3. The fusion protein of claim 1, wherein the GDF15 region and the Fcdomain are joined by a polypeptide linker.
 4. The fusion protein ofclaim 3, wherein the polypeptide linker comprises the amino acidsequence of SEQ ID NO: 18, 30, 34, 40, 58, 61, 64, 69, 72, 75, 78, 113,116, 119, 122, 125, or
 128. 5. The fusion protein of claim 1, whereinthe fusion protein comprises the amino acid sequence of SEQ ID NO: 222,225, 229, or
 232. 6. The fusion protein of claim 1, wherein the fusionprotein comprises two or more Fc domains.
 7. A dimer comprising (i) afirst polypeptide chain comprising a GDF15 region and a first Fc domain,wherein the Fc domain comprises the amino acid sequence of SEQ ID NO:220 or SEQ ID NO: 220 with a tyrosine to cysteine mutation at position113 and (ii) a second polypeptide chain comprising a second Fc domain.8. The dimer of claim 7, wherein second Fc domain comprises the aminoacid sequence of SEQ ID NO: 16, 22, 28, 29, 33, 35, 38, 48, 85, 91, 106,132, 141, 148, 155, 162, 169, 176, 183, 192, 199, 206, 213, 220, 227,233, 236, 268, 275, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290,291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301 or
 302. 9. Thedimer of claim 7, wherein the GDF15 region comprises the amino acidsequence of SEQ ID NO: 4, 8, 12, 25, 52 and
 55. 10. The dimer of claim7, wherein the GDF15 region and the first Fc domain are joined by apolypeptide linker.
 11. The dimer of 10, wherein the polypeptide linkercomprises the amino acid sequence of SEQ ID NO: 18, 30, 34, 40, 58, 61,64, 69, 72, 75, 78, 113, 116, 119, 122, 125, or
 128. 12. The dimer ofclaim 7, wherein the first polypeptide comprises the amino acid sequenceof SEQ ID NO: 222, 225, 229, or
 232. 13. The dimer of claim 7, whereinthe first and second polypeptide chains are non-covalently associated.14. The dimer of claim 7, wherein the first and second polypeptidechains are covalently associated.
 15. The dimer of claim 14, wherein thefirst and second polypeptide chains are covalently associated viadisulfide bonds between their respective Fc domains.
 16. A tetramercomprising (i) a first dimer and (ii) a second dimer, wherein the firstand second dimer independently comprise a dimer comprising (a) a firstpolypeptide chain comprising a GDF15 region and a first Fc domain,wherein the first Fc domain comprises the amino acid sequence of SEQ IDNO: 220 or SEQ ID NO: 220 with a tyrosine to cysteine mutation atposition 113, and (b) a second polypeptide chain comprising a second Fcdomain, and wherein the first polypeptide chain of the first dimer islinked to the first polypeptide chain of the second dimer via aninterchain disulfide bond between their respective GDF15 regions. 17.The tetramer of claim 16, wherein the second Fc domain of the firstdimer, of the second dimer, or of both the first and second dimerscomprise the amino acid sequence of SEQ ID NO: 16, 22, 28, 29, 33, 35,38, 48, 85, 91, 106, 132, 141, 148, 155, 162, 169, 176, 183, 192, 199,206, 213, 220, 227, 233, 236, 268, 275, 281, 282, 283, 284, 285, 286,287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300,301 or
 302. 18. The tetramer of claim 16, wherein the GDF15 region ofthe first polypeptide chain of the first dimer comprises the amino acidsequence of SEQ ID NO: 4, 8, 12, 25, 52 or
 55. 19. The tetramer of claim16, wherein the GDF15 region and the first Fc domain of the first dimer,of the second dimer, or of both the first and second dimers are joinedby a polypeptide linker.
 20. The tetramer of claim 19, wherein thepolypeptide linker comprises the amino acid sequence of SEQ ID NO: 18,30, 34, 40, 58, 61, 64, 69, 72, 75, 78, 113, 116, 119, 122, 125, or 128.21. The tetramer of claim 16, wherein the first polypeptide of the firstdimer comprises the amino acid sequence of SEQ ID NO: 222, 225, 229, or232.
 22. The tetramer of claim 16, wherein first polypeptide of thesecond dimer comprises the amino acid sequence of SEQ ID NO: 222, 225,229, or
 232. 23. The fusion protein of claim 1, wherein the GDF15 regioncomprises an amino acid sequence that is at least about 85 percentidentical to SEQ ID NO: 4, 8, or
 12. 24. The fusion protein of claim 23,wherein the GDF15 region comprises an amino acid sequence that is atleast about 85 percent identical to SEQ ID NO: 12 and comprises amutation at position
 3. 25. The fusion protein of claim 24, wherein themutation at position 3 is a N3Q mutation.
 26. The fusion protein ofclaim 25, wherein the GDF15 region and the Fc domain are joined by apolypeptide linker.
 27. The fusion protein of claim 26, wherein thepolypeptide linker comprises the amino acid sequence of SEQ ID NO: 18,30, 34, 40, 58, 61, 64, 69, 72, 75, 78, 113, 116, 119, 122, 125, or 128.